Xenotropic Murine Leukemia Virus-related Virus Research Articles

False Alarm: XMRV, Cancer, and Chronic Fatigue Syndrome.

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) was first described in 2006 in some human prostate cancers. But it drew little attention until 2009, when it was also found, as infectious virus and as MLV-related DNA, in samples from people suffering from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). This discovery was rapidly followed by efforts of the international research community to understand the significance of the association and its potential to spread widely as an important human pathogen. Within a few years, efforts by researchers worldwide failed to repeat these findings, and mounting evidence for laboratory contamination with mouse-derived virus and viral DNA sequences became accepted as the explanation for the initial findings. As researchers engaged in these studies, we present here a historical review of the rise and fall of XMRV as a human pathogen, and we discuss the lessons learned from these events.

A Novel Pathogen of an Emerging Infectious Disease (Large Kidney Disease) in Farmed Blue Foxes

An emerging infectious disease (EID) in foxes called “large kidney disease”, characterized by enlarged kidneys, has been breaking out in fox farms in China, although its pathogenesis has not yet been elucidated. Here, we performed viral metagenomics sequencing on diseased fox tissue samples that identified a virus with 82.6% homology to the xenotropic murine leukemia virus-related virus (XMRV) PreXMRV-1 provirus strain (GenBank accession number NC_007815.2) in sick fox tissue. It was named PreXMRV-20, and its genome was verified by reverse transcription-polymerase chain reaction (PCR) and PCR product sequencing. Nonenveloped and polygonal virus-like particles consistent with the shape and size of XMRV were observed by negative staining electron microscopy. Administered subcutaneously, PreXMRV-20 infected weaned foxes, leading to growth retardation. The discovery of the PreXMRV-20 strain (the first isolation of an XMRV homolog in blue foxes) identifies a potential public health issue in blue fox breeding since XMRV has been confirmed to be a zoonotic virus.

Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification.

In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log10 reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65-3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.

Continuous low pH viral inactivation: Operation and scaling strategy informs viral clearance study.

A continuous viral inactivation (CVI) tubular reactor was designed for low pH viral inactivation within a continuous downstream system across multiple scales of operation. The reactors were designed to provide a minimum residence time of >60 min. The efficacy of this tubular reactor was tested with xenotropic murine leukemia virus (X-MuLV)through pulse injection experiments. It was determined that the minimum residence time of the small-scale reactor design, when operated at the target process flow rate, occurred between 63 and 67 min. Inactivation kinetics were compared between continuous operation and standard batch practices using three monoclonal antibodies. The quantification of the virus log reduction values (LRV) wassimilar between the two modes of operation and most of the acid-treated samples had virus concentrations below the limit of detection. However, residual infectivity was still present in the endpoint batch samples of two experiments while the continuous samples always remained below the limit of detection. This provides the foundation for leveraging a standard batch-based model to quantify the LRV for a CVI unit operation.

Development of a transient inline spiking system for evaluating virus clearance in continuous bioprocessing-Proof of concept for virus filtration.

The development of continuous/connected bioprocesses requires new approaches for viral clearance validation, both for specific unit operations and for the overall process. In this study, we have developed a transient inline spiking system that can be used to evaluate virus clearance at distinct time points during prolonged operation of continuous bioprocesses. The proof of concept for this system was demonstrated by evaluating the viral clearance for a virus filtration step, both with and without a prefilter upstream of the virus filter. The residence time distribution was evaluated using a previously identified noninteracting fluorescent tracer, while viral clearance was evaluated from measurements of the virus titer in samples obtained downstream of the virus filter. The measured log reduction values (LRV) for ϕX174, minute virus of mice, xenotropic murine leukemia virus, and a noninfectious mock virus particlewere all within 0.5 log of those obtained using a traditional batch virus challenge for both model and real-world process streams (LRV between 2.2 and 3.4 for ϕX174 using a single layer of virus filter). The results demonstrate the effectiveness of transient inline spiking to validate the virus clearance capabilities in continuous bioprocessing, an essential element for the adoption of these processes for products made using mammalian cell lines.

Оценка эффективности удаления и инактивации вирусов в модельном процессе производства биотехнологического лекарственного препарата на основе рекомбинантного Fab-фрагмента моноклонального антитела

Virus safety insuring is one of the most serious problems in the development of biotechnological drugs produced using animal cell lines. Quantitative assessment of virus removal and inactivation is an integral approach to show the reliability of the target compound production. In this work, the model experiments have been carried out on the purification of the monoclonal antibody recombinant Fab-fragment from viruses of various origins and properties: xenotropic murine leukemia virus (X-MuLV), pseudorabies virus (PRV), Reo-3 virus and encephalomyocarditis virus (EMCV). It was shown that two methods, acidification to pH 3.2 and nanofiltration, made it possible to reduce the virus infectivity to levels undetectable in cell cultures (according to TCID50). The developed multistage purification process of the target protein provided an overall decrease in viral clearance to the following values: X-MuLV≥10.17lg, PRV≥13.98lg, Reo-3≥8.09lg and EMCV≥4.98lg. These results confirm that the developed technology ensures the virus safety during the production of a monoclonal antibody recombinant Fab-fragment by CHO cell line. These results confirm virus safety of production technology of recombinant monoclonal antibody Fab-fragment produced in CHO cell line. virus safety, virus elimination, virus inactivation, nanofiltration, Fab-fragment, monoclonal antibody, CHO cell line

Characterization of a new case of XMLV (Bxv1) contamination in the human cell line Hep2 (clone 2B)

The use of misidentified cell lines contaminated by other cell lines and/or microorganisms has generated much confusion in the scientific literature. Detailed characterization of such contaminations is therefore crucial to avoid misinterpretation and ensure robustness and reproducibility of research. Here we use DNA-seq data produced in our lab to first confirm that the Hep2 (clone 2B) cell line (Sigma-Aldrich catalog number: 85011412-1VL) is indistinguishable from the HeLa cell line by mapping integrations of the human papillomavirus 18 (HPV18) at their expected loci on chromosome 8. We then show that the cell line is also contaminated by a xenotropic murine leukemia virus (XMLV) that is nearly identical to the mouse Bxv1 provirus and we characterize one Bxv1 provirus, located in the second intron of the pseudouridylate synthase 1 (PUS1) gene. Using an RNA-seq dataset, we confirm the high expression of the E6 and E7 HPV18 oncogenes, show that the entire Bxv1 genome is moderately expressed, and retrieve a Bxv1 splicing event favouring expression of the env gene. Hep2 (clone 2B) is the fourth human cell line so far known to be contaminated by the Bxv1 XMLV. This contamination has to be taken into account when using the cell line in future experiments.

Identification and characterization of a Triton X‐100 replacement for virus inactivation

Triton X-100 detergent treatment is a robust enveloped virus inactivation unit operation included in biopharmaceutical manufacturing processes. However, the European Commission officially placed Triton X-100 on the Annex XIV authorization list in 2017 because a degradation product of Triton X-100, 4-(1,1,3,3-tetramethylbutyl) phenol (also known as 4-tert-octylphenol), is considered to have harmful endocrine disrupting activities. As a result, the use of Triton X-100 in the European Economic Area (EEA) would not be allowed unless an ECHA issued authorization was granted after the sunset date of January 4, 2021. This has prompted biopharmaceutical manufacturers to search for novel, environment-friendly alternative detergents for enveloped virus inactivation. In this study, we report the identification of such a novel detergent, Simulsol SL 11W. Simulsol SL 11W is an undecyl glycoside surfactant produced from glucose and C11 fatty alcohol. We report here that Simulsol SL 11W was able to effectively inactive enveloped viruses, such as xenotropic murine leukemia virus (XMuLV) and pseudorabies virus (PRV). By using XMuLV as a representative enveloped virus, the influence of various parameters on the effectiveness of virus inactivation was evaluated. Virus inactivation by Simulsol SL 11W was effective across different clarified bioreactor harvests at broad concentrations, pH, and temperature ranges. Simulsol SL 11W concentration, temperature of inactivation, and treatment time were identified as critical process parameters for virus inactivation. Removal of Simulsol SL 11W was readily achieved by Protein A chromatography and product quality was not affected by detergent treatment. Taken together, these results have shown the potential of Simulsol SL 11W as a desirable alternative to Triton X-100 for enveloped virus inactivation that could be readily implemented into biopharmaceutical manufacturing processes.

Truly continuous low pH viral inactivation for biopharmaceutical process integration.

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities—which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry‐standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system‐induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.

Insights into virus inactivation by polysorbate 80 in the absence of solvent.

Triton X-100 has long been used either alone or in combination with solvent to inactivate enveloped viruses in biopharmaceutical manufacturing. However, European Chemicals Agency (ECHA) officially placed Triton X-100 on the Annex XIV authorization list in 2017 because 4-(1,1,3,3-tetramethylbutyl) phenol, a degradation product of Triton X-100, is of harmful endocrine disrupting activities. As a result, any use of Triton X-100 in the European Economic Area would require an ECHA issued authorization after the sunset date of January 4, 2021. In search of possible replacements for Triton X-100, we discovered that polysorbate 80 (PS80) in absence of any solvents was able to effectively inactive enveloped viruses such as xenotropic murine leukemia virus and pseudorabies virus with comparable efficacy as measured by log reduction factors. Interestingly, PS80 did not show any virucidal activities in phosphate buffered saline (PBS) while achieving robust virus inactivation in cell-free Chinese hamster ovary (CHO) bioreactor harvests. This intriguing observation led us to speculate that virus inactivation by PS80 involved components in the cell-free CHO bioreactor harvests that were absent in PBS. Specifically, we hypothesized that esterase and/or lipases in the cell-free bioreactor harvests hydrolyzed PS80 to yield oleic acid, a known potent virucidal agent, which in turn inactivated viruses. This theory was confirmed using purified recombinant lysosomal phospholipase A2 isomer (rLPLA2) in PBS. Subsequent characterization work has indicated that virus inactivation by PS80 is effective and robust within temperature and concentration ranges comparable to those of Triton X-100. Similar to Triton X-100, virus inactivation by PS80 is dually dependent on treatment time and temperature. Unlike Triton X-100, PS80 inactivation does not correlate with concentrations in a simple manner. Additionally, we have demonstrated that PS20 exhibits similar virus inactivation activities as PS80. Based on the findings described in the current work, we believe that PS80 is potentially a viable replacement for Triton X-100 and can be used in manufacturing processes for wide spectrum of biopharmaceuticals to achieve desirable virus clearance. Finally, the advantages and disadvantages of using PS80 for virus inactivation are discussed in the contexts of GMP manufacturing.

Virus removal robustness of ion exchange chromatography

Virus removal by ion exchange chromatography enhances the safety profile of therapeutic protein products. The robustness of virus removal depends on electrostatic binding between virus and oppositely charged chromatography media. However, model retrovirus Xenotropic Murine Leukemia Virus (XMuLV) binding remains robust even when virus and media are both positively charged. We investigated this counter-intuitive phenomenon using side-by-side comparison of virus-media binding behavior of XMuLV versus parvovirus, two viruses very different in size and structure but comparable in isoelectric point. When both viruses were negatively charged, XMuLV bound to positive anion exchange media with higher strength than parvovirus. When both viruses were positively charged, XMuLV remained tightly bound to positive media but parvovirus was dissociated. Likewise, XMuLV binding to media was much stronger than parvovirus under cation exchange conditions. These findings suggest that XMuLV binding could be enhanced by localized charge distribution not possessed by parvovirus, which is an important consideration for designing chromatography processes with robust virus removal capacity.

Characterizing and enhancing virus removal by protein A chromatography.

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.

Virus study for continuous low pH viral inactivation inside a coiled flow inverter.

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.

A Survey on Human T-cell Lymphotropic Virus Type 1 (HTLV-1) and Xenotropic Murine Leukemia Virus-Related Virus (XMRV) Coinfection in Tehran, Iran.

Background:Xenotropic murine leukemia virus-related virus (XMRV) is a gamma retrovirus, which has been detected in patients with prostate cancer, chronic fatigue syndrome, and general population with a number of acquired infections such as infection with human T-cell lymphotropic virus (HTLV) and human immunodeficiency virus (HIV). The aim of this study was to determine the HTLV-1 and XMRV coinfection for the first time in Iranian patients who were admitted to the Tehran hospitals.Materials and Methods:Two hundred and ninety one patients suspected with HTLV-1 were referred to the hospitals affiliated to the Iran University of Medical Sciences, Tehran, Iran from April 2012 to October 2016. Genomic deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) from peripheral blood mononuclear cells/cerebrospinal fluids was extracted by High Pure Viral Nucleic Acid Kit (Roche, Germany). After complementary DNA synthesis, conventional reverse transcriptase polymerase chain reaction was used for the detection of HTLV-1 or XMRV-infected patients. Statistical Package for Social Sciences (SPSS) software, version 16 (SPSS, Chicago, IL, USA) was used for statistical analyses.Results:Of the 291 patients suspected of HTLV infection, 123 (42.3%) were male with a mean age of 38±15 years. HTLV-1 RNA was found in 93 (31.9%) specimens comprising 40 men (41.3%) and 53 women (56.9%). Of the 93 patients who were HTLV-1 positive, one sample (1%) was positive for XMRV env gene.Conclusion:These findings suggest that the lack of significant detection of XMRV in patients who were HTLV-1 positive could not be associated with complications of HTLV-1. Although this is a preliminary report from Iranian patients with HTLV-1, further studies are needed to show the actual prevalence of XMRV infection by geographical distribution and various populations.

Elimination of Mycoplasma Contamination from Infected Human Hepatocyte C3A Cells by Intraperitoneal Injection in BALB/c Mice.

Background/Aims: The use of antibiotics to eliminate Mycoplasma contamination has some serious limitations. Mycoplasma contamination can be eliminated by intraperitoneal injection of BALB/c mice with contaminated cells combined with screening monoclonal cells. However, in vivo passage in mice after injection with contaminated cells requires a long duration (20–54 days). Furthermore, it is important to monitor for cross-contamination of mouse and human cells, xenotropic murine leukemia virus-related virus (XMRV) infection, and altered cell function after the in vivo treatment. The present study aimed to validate a reliable and simplified method to eliminate mycoplasma contamination from human hepatocytes. BALB/c mice were injected with paraffin oil prior to injection with cells, in order to shorten duration of intraperitoneal passage. Cross-contamination of mouse and human cells, XMRV infection and cell function-related genes and proteins were also evaluated.Methods: PCR and DNA sequencing were used to confirm Mycoplasma hyorhinis (M. hyorhinis) contamination in human hepatocyte C3A cells. Five BALB/c mice were intraperitoneally injected with 0.5 ml paraffin oil 1 week before injection of the cells. The mice were then intraperitoneally injected with C3A hepatocytes (5.0 × 106/ml) contaminated with M. hyorhinis (6.2 ± 2.2 × 108 CFU/ml). Ascites were collected for monoclonal cell screening on the 14th day after injection of contaminated cells. Elimination of mycoplasma from cells was determined by PCR and Transmission Electron Microscopy (TEM). Human–mouse cell and XMRV contamination were also detected by PCR. Quantitative reverse transcription PCR and western blotting were used to compare the expression of genes and proteins among treated cells, non-treated infected cells, and uninfected cells.Results: Fourteen days after injection with cells, 4 of the 5 mice had ascites. Hepatocyte colonies extracted from the ascites of four mice were all mycoplasma-free. There was no cell cross-contamination or XMRV infection in treated cell cultures. Elimination of Mycoplasma resulted in partial or complete recovery in the expression of ALB, TF, and CYP3A4 genes as well as proteins. Proliferation of the treated cells was not significantly affected by this management.Conclusion: The method of elimination of Mycoplasma contamination in this study was validated and reproducible. Success was achieved in four of five cases examined. Compared to the previous studies, the duration of intraperitoneal passage in this study was significantly shorter.

Ductal variant of prostate adenocarcinoma harbor Xenotropic murine leukemia virus related virus (XMRV) infection: a novel finding in subtype of prostate cancer.

Xenotropic murine leukemia virus related virus (XMRV), is the first gammaretrovirus identified a decade ago, in human tissue bearing adenocarcinoma of prostate, followed by several researches documenting little or no prevalence of XMRV in prostate cancer samples. However, the status of XMRV within subtype of prostate adenocarcinoma has not been investigated yet. In this study, we investigated the relationship between XMRV and broad spectrum morphological entities of prostate adenocarcinoma, including acinar, ductal and other rare subtypes. The prevalence of XMRV DNA in different histological subtypes of prostate adenocarcinoma was examined after characterizing the tumors into groups, using formalin-fixed, paraffin-embedded tissue samples from newly diagnosed prostate adenocarcinomas and archival prostate cancer tissue from our XMRV case control analysis. Broad-spectrum XMRV DNA amplification was performed by end-point polymerase chain reaction, using commercially available primer set. The study included 100 patients with prostate cancer. XMRV DNA was detected in 4 of 8 (50%) ductal adenocarcinomas, exhibiting papillary and cribriform histological features. XMRV DNA was not detected in any other variant of adenocarcinoma including acinar (0/91) and mucinous carcinomas (0/1). Majority of XMRV positive cases were biologically aggressive and present cancer at an early age upon diagnosis. Ductal adenocarcinomas demonstrate a significant association of XMRV DNA while other histological variants of prostate adenocarcinoma seem unrelated to XMRV infection.

Problems with precaution: the transfusion medicine experience

The precautionary principle is a dominant paradigm governing risk-based decision-making. Today, there are increasing pressures to re-examine aggressive precautionary approaches, and to assess how the principle should be applied in the modern system. In this paper, we examined three key applications of precautionary approaches in the field of transfusion medicine to provide insight into the risks and benefits of these approaches. The three case studies examined were the donor deferral policies to safeguard against transfusion transmission of human immunodeficiency virus, variant Creutzfeldt–Jacob disease, and, lastly, xenotropic murine leukemia virus-related virus. Characterization of precautionary applications was conducted using an embedded case study design. Our findings indicate that transfusion transmission mitigation strategies have become increasingly aggressive in the face of theoretical risks. In contrast, the review processes for implementation and reversal of precautionary policies have been slow, and historical donor deferral policies are still in place today. Application of precautionary approaches has proved challenging with both benefits and pitfalls. In light of emerging threats to the blood system, policy-makers should consider the implementation of frameworks to guide the appropriate application of precaution in transfusion medicine in the future.

XMRV and Public Health: The Retroviral Genome Is Not a Suitable Template for Diagnostic PCR, and Its Association with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Appears Unreliable

A few years ago, a highly significant association between the xenotropic murine leukemia virus-related virus (XMRV) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a complex debilitating disease of poorly understood etiology and no definite treatment, was reported in Science, raising concern for public welfare. Successively, the failure to reproduce these findings, and the suspect that the diagnostic PCR was vitiated by laboratory contaminations, led to the retraction of the paper. Notwithstanding, XMRV continued to be the subject of researches and public debates. Occasional positivity in humans was also detected recently, even if the data always appeared elusive and non-reproducible. In this study, we discuss the current status of this controversial association and propose that a major role in the unreliability of the results was played by the XMRV genomic composition in itself. In this regard, we present bioinformatic analyses that show: (i) aspecific, spurious annealings of the available primers in multiple homologous sites of the human genome; (ii) strict homologies between whole XMRV genome and interspersed repetitive elements widespread in mammalian genomes. To further detail this scenario, we screen several human and mammalian samples by using both published and newly designed primers. The experimental data confirm that available primers are far from being selective and specific. In conclusion, the occurrence of highly conserved, repeated DNA sequences in the XMRV genome deeply undermines the reliability of diagnostic PCRs by leading to artifactual and spurious amplifications. Together with all the other evidences, this makes the association between the XMRV retrovirus and CFS totally unreliable.

Detection of Viruses in Human Cell Lines Applying Next Generation Sequencing

It is widely accepted that diverse viral infections can at least contribute to the development of tumor cells in various ways. The viruses can directly alter the eukaryotic genome by DNA integration, alter the gene regulation, or may cause a chronic inflammation. Viral infections can either be lytic causing the production of new viruses or latent with no viral replication. However, in both cases various viral genes are constantly transcribed in the eukaryotic cells. Next generation sequencing (NGS) offers the possibility to capture the whole transcriptome of the cells via RNA-seq including host and viral mRNA. Over the years, we have detected human pathogenic viruses as well as other viruses potentially infecting human cell lines from our cell lines collection applying PCR or RT-PCR. The viruses comprise Epstein-Barr-virus (EBV; human herpesvirus type 4), hepatitis B virus (HBV), hepatitis C virus (HCV), human herpesvirus type 8 (HHV-8), human immunodeficiency virus type 1 and 2 (HIV-1/-2), human papilloma virus (HPV), human T-lymphotropic virus type 1 and 2 (HTLV-1/-2), squirrel monkey retrovirus (SMRV), xenotropic murine leukemia virus (XMLV; including xenotropic murine leukemia virus related virus, XMRV). These data were now compared with the results obtained from the evaluation of RNA-seq and whole exome sequencing (WES) data of the Cancer Cell Lines Encyclopedia (CCLE). We screened 133 RNA seq and 62 WES datasets of the CCLE sequence database for the presence of the previously mentioned viral sequences. NCBI reference complete genome sequences of the respective viruses and the human hg38 genome were used to for the alignment. In these two datasets 118 and 58 cell lines were leukemia/lymphoma cell lines, respectively, comprising the different hematopoietic lineages. Eleven B-cell derived cell lines were concordantly EBV positive in PCR analysis and in RNA-seq. The DOHH-2 cell line exhibited a relatively low number of alignments. This is concordant with our finding applying fluorescent in situ hybridization that this cell line consists of two clones: one infected with EBV and one EBV-free clone. Both clones could be separated by single cell cloning. Comparing RNA-seq and WES, RNA-seq revealed more virus specific reads relative to the total reads (max. 0.5425% vs. max. 0.0026%). Thus, RNA-seq appears to be more sensitive than WES. HHV-8 was concordantly clearly detected by PCR and RNA-seq as well as SMRV in the NAMALWA cell line. To further evaluate the robustness of the virus detection method, we included some viruses not specific for hematopoietic cells, but shown to be positive in distinct cell lines applying PCR: HBV and XMLV. We found complete concordance between PCR and RNA-seq in two liver cell lines (HEP-3B and SNU-886) and - except for the melanoma cell line SK-MEL-1 - XMLV was also detected in PCR positive cell lines by RNA-seq (three hematopoietic cell lines and six non-hematopoietic cell lines). Concerning the SK-MEL-1 cell line, different subcultures of the cell line might have been tested with the two methods, one subculture after heterotransplantation into rodents with PCR assay and the RNA-seq negative one originating from a subclone without previous heterotransplantation. Taken together, only one out of 21 virus positive cell lines were discordant applying RNA-seq. On the other hand, all PCR-negative cell lines were also negative by RNA-seq. Significant background alignments in the range of 0 to 850 reads could be detected only with the retroviruses XMLV, SMRV and HTLV-1 regarding RNA-seq, whereas the positive samples were all above 1x105. The background alignments might be attributed to some homologue sequences to endogenous retroviral elements in the human genome. In summary, RNA-seq can be used as reliable and single-step method to analyze simultaneously a panel of potential virus infections in cell cultures and thereby delivering additional viral information beside host gene expression. Future studies might demonstrate whether non-human mappable reads in RNA-seq data could be used to detect new viruses infecting human cells and being potentially implicated in tumor formation. DisclosuresNo relevant conflicts of interest to declare.

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins.

ABSTRACTGammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes.