Identification of XMRV Infection-Associated microRNAs in Four Cell Types in Culture

INTRODUCTION: XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture. METHODS: Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray. RESULTS: MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types. DISCUSSION: The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types

Susceptibility of human primary neuronal cells to Xenotropic Murine Leukemia Virus-related (XMRV) virus infection

<p>Abstract</p> <p>Background</p> <p>Xenotropic Murine Leukemia Virus-related (XMRV) virus is a recently identified mouse gammaretrovirus that has the ability to infect certain human cells. In this study, we investigated the susceptibility of primary neuronal cell types to infection with XMRV.</p> <p>Findings</p> <p>We observed that the human primary progenitors, progenitor-derived neurons, and progenitor-derived astrocytes supported XMRV multiplication. Interestingly, both progenitors and progenitor-derived neurons were more susceptible compared with progenitor-derived astrocytes. In addition, XMRV-infected Jurkat cells were able to transmit infection to neuronal cells.</p> <p>Conclusions</p> <p>These data suggest that neuronal cells are susceptible for XMRV infection.</p

Enhanced diagnostic efficiency of the polymerase chain reaction by co-amplification of multiple regions of HIV-1 and HIV-2

A method for co-amplification of multiple viral sequences of HIV-1 and HIV-2 by polymerase chain reaction was designed. The technique resulted in the specific detection of each type of virus and allowed the amplification of as few as two copies of target DNA. The amplification of multiple regions of the viral genome offers the advantage of detecting multiple target sequences, which may be essential for some viruses, such as HIV, that exhibit a high degree of variability in their gene sequences. In addition, the method permitted the detection of both virus types in the same reaction, allowing for greater utility in geographic areas where coinfections with both viruses occur and cross-reactivity in Western blots is observed. This method was applied successfully to the detection of viral DNA in clinical specimens.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31395/1/0000309.pd

Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay

<p>Abstract</p> <p>Background</p> <p>For more than a decade there has been increasing interest in the use of nanotechnology and microarray platforms for diagnostic applications. In this report, we describe a rapid and simple gold nanoparticle (NP)-based genomic microarray assay for specific identification of avian influenza virus H5N1 and its discrimination from other major influenza A virus strains (H1N1, H3N2).</p> <p>Results</p> <p>Capture and intermediate oligonucleotides were designed based on the consensus sequences of the matrix (M) gene of H1N1, H3N2 and H5N1 viruses, and sequences specific for the hemaglutinin (HA) and neuraminidase (NA) genes of the H5N1 virus. Viral RNA was detected within 2.5 hours using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining in the absence of RNA fragmentation, target amplification, and enzymatic reactions. The lower limit of detection (LOD) of the assay was less than 100 fM for purified PCR fragments and 10³TCID₅₀units for H5N1 viral RNA.</p> <p>Conclusions</p> <p>The NP-based microarray assay was able to detect and distinguish H5N1 sequences from those of major influenza A viruses (H1N1, H3N2). The new method described here may be useful for simultaneous detection and subtyping of major influenza A viruses.</p

Absence of Detectable XMRV and Other MLV-Related Viruses in Healthy Blood Donors in the United States

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

XMRV: usage of receptors and potential co-receptors

<p>Abstract</p> <p>Background</p> <p>XMRV is a gammaretrovirus first identified in prostate tissues of Prostate Cancer (PC) patients and later in the blood cells of patients with Chronic Fatigue Syndrome (CFS). Although XMRV is thought to use XPR1 for cell entry, it infects A549 cells that do not express XPR1, suggesting usage of other receptors or co-receptors.</p> <p>Methods</p> <p>To study the usage of different receptors and co- receptors that could play a role in XMRV infection of lymphoid cells and GHOST (GFP- Human osteosarcoma) cells expressing CD4 along with different chemokine receptors including CCR1, CCR2, etc., were infected with XMRV. Culture supernatants and cells were tested for XMRV replication using real time quantitative PCR.</p> <p>Results</p> <p>Infection and replication of XMRV was seen in a variety of GHOST cells, LNCaP, DU145, A549 and Caski cell lines. The levels of XMRV replication varied in different cell lines showing differential replication in different cell lines. However, replication in A549 which lacks XPR1 expression was relatively higher than DU145 but lower than, LNCaP. XMRV replication varied in GHOST cell lines expressing CD4 and each of the co- receptors CCR1-CCR8 and bob. There was significant replication of XMRV in CCR3 and Bonzo although it is much lower when compared to DU145, A549 and LNCaP.</p> <p>Conclusion</p> <p>XMRV replication was observed in GHOST cells that express CD4 and each of the chemokine receptors ranging from CCR1- CCR8 and BOB suggesting that infectivity in hematopoietic cells could be mediated by use of these receptors.</p