Characterisation and functional analysis of the murine gammaherpesvirus-68-encoded microRNAs

All mammalian cells encode microRNAs (miRNAs), which are small non-coding RNAs (~ 22 nucleotides) that control numerous physiological processes via regulation of gene expression. A number of viruses, in particular herpesviruses, also encode miRNAs. Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi’s sarcoma associated herpesvirus (KSHV) are associated with lymphoproliferative disorders and some types of cancer in humans. Gammaherpesvirus-encoded miRNAs are predicted to contribute to pathogenesis and virus life cycle by suppressing host and viral target genes. However, the exact functions of these miRNAs during virus infection in the natural host are largely unknown. Strict species specificity has limited research on the human gammaherpesviruses mainly to in vitro studies. Murine gammaherpesvirus 68 (MHV-68) encodes at least 15 miRNAs and provides a unique tractable small animal model to investigate in vivo gammaherpesvirus pathogenic features that are difficult to assess in humans. Following intranasal infection of lab mice, the virus undergoes primary lytic infection in the lung epithelial cells and then spreads to the spleen establishing latent infection in splenic B lymphocytes, macrophages, and dendritic cells. The peak of the latent viral load occurs in the spleen at 14 dpi and then it decreases over time, but the virus is not completely eliminated and the latent viral genomes remain in the host cells for lifetime and can reactivate to produce infectious virus under certain conditions. The aims of my project were to: (1) establish and develop quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for quantification of the MHV-68 miRNAs, (2) determine the miRNAs expression profiles during the two stages of virus infection (lytic and latent infection), (3) investigate the kinetics of the miRNAs expression during latency in vivo, (4) construct an MHV-68 miRNA mutant virus lacking 9 miRNAs (designated MHV-68.ΔmiRNAs), and (5) carry out thorough phenotypic characterisation of this mutant virus in order to determine the possible functions MHV-68 miRNAs in the context of natural host infection. It was found that the MHV-68 miRNAs expression pattern varied during different stages of infection, suggesting a differential regulation of the expression of these miRNAs depending on the phase of infection. In order to investigate the kinetics of miRNAs expression during latency in vivo, BALB/c mice were infected intranasally with MHV- 68 virus and spleens were harvested at days 10, 14, 21, and 32 post infection. The levels of miRNAs expression were determined by qRT-PCR in the splenocytes from infected mice. Interestingly, in contrast to the lytic MHV-68 protein coding genes, the expression of the miRNAs increased over time after 21 dpi, suggesting that the MHV-68-encoded miRNAs may play more fundamental roles during later stages of latent infection. In order to determine the potential roles of the MHV-68 miRNAs in virus pathogenesis, a miRNA mutant virus lacking the expression of 9 miRNAs, named MHV- 68.ΔmiRNAs, was constructed. The miRNA mutant virus replicated with the same kinetics as wild type virus in vitro and in vivo demonstrating that the deleted MHV-68 miRNAs are dispensable for virus lytic replication. To examine the roles of the miRNAs during virus latency, the MHV-68.ΔmiRNAs virus was characterised throughout a 49- day course of infection. Although the level of ex vivo reactivation of the MHV-68.ΔmiRNAs virus was comparable to that of the WT virus during the establishment of latency and as late as 28 dpi, the reactivation of the MHV-68.ΔmiRNAs virus was approximately 18-times higher than that of the WT virus at 49 dpi despite the similar levels of the genomic viral DNA loads at the same time-point. This suggests that the MHV-68 miRNAs suppress virus reactivation and promote maintenance of long-term latency. Moreover, the lytic viral gene expression levels were higher in splenocytes from the MHV-68.ΔmiRNAs-infected mice than the basal expression levels in the splenocytes from WT MHV-68-infected mice, suggesting that the MHV-68 miRNAs may suppress viral lytic gene expression during long-term latency in vivo and thus help the virus lay low

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells

Titrated Misoprostol Versus Dinoprostone for Labor Induction

Background: Misoprostol is as effective as dinoprostone for labor induction with low cost and temperature stability.Aim: This study designed to compare titrated misoprostol regarding its safety and efficacy with dinoprostone for induction of labor.Subjects and Methods: Women with a single pregnancy, above 37 weeks’ gestation, cephalic presentation, modified Bishop’s score <8, and not in labor with reassuring fetal heart rate, admitted for labor induction enrolled in this randomized controlled study. Studied women were randomized into; Group I: received oral misoprostol titrated in sterile water (200 μg tablet was dissolved in 200 ml sterile water [1 μg/ml]), starting dose of 20 μg misoprostol required, given every 2 h, and stopped if adequate contractions obtained and Group II: received vaginal dinoprostone tablet maximum two doses followed by augmentation of labor by oxytocin ± amniotomy if there is no uterine contractions after two doses of dinoprostone. In Group I, if the contractions were inadequate after two doses of oral titrated misoprostol (20 μg [20 ml]), the starting dose increased to 40 μg (40 ml), escalating the dose from 5 to 10 ml (45–50 μg), and 20 ml (60 μg) maximum ± amniotomy. If the uterine contractions were adequate, the next dose of misoprostol or dinoprostone was omitted. Statistical analysis done using Student’s t‑test for quantitative data and Chi‑square test for qualitative data.Results: Induction‑to‑delivery time was significantly longer in misoprostol than dinoprostone group (975 vs. 670 min, respectively), (P = 0.01). About 20.2% (21/104) of women in misoprostol group did not deliver vaginally within 24 h compared to 7.4% (8/108) in dinoprostone group (significant difference, P = 0.01). Augmentation of labor was significantly high in dinoprostone (37.96% [41/108]) compared to misoprostol group (10.6% [11/104]) (P < 0.01).Conclusion: Titrated misoprostol for induction of labor seems to be associated with significantly longer induction‑to‑delivery time, low incidence of vaginal birth within 24 h, and less need for augmentation of labor compared to vaginal dinoprostone.KEY WORDS: Dinoprostone, labor induction, titrated misoprosto

Epstein-Barr Virus genome deletions in Epstein-Barr Virus-positive T/NK cell lymphoproliferative diseases

The main target cells for Epstein-Barr virus (EBV) infection and persistence are B lymphocytes, although T and NK cells can also become infected. In this paper, we characterize the EBV present in 21 pediatric and adult patients who were treated in France for a range of diseases that involve infection of T or NK cells. Of these 21 cases, 5 pediatric patients (21%) and 11 adult patients (52%) were of Caucasian origin. In about 30% of the cases, some of the EBV genomes contain a large deletion. The deletions are different in every patient but tend to cluster near the BART region of the viral genome. Detailed investigation of a family in which several members have persistent T or NK cell infection by EBV indicates that the virus genome deletions arise or are selected independently in each individual patient. Genome sequence polymorphisms in the EBV in these T or NK cell diseases reflect the geographic origin of the patient and not a distinct type of EBV (the 21 cases studied included examples of both type 1 and type 2 EBV infection). Using virus produced from type 1 or type 2 EBV genomes cloned in bacterial artificial chromosome (BAC) vectors, we demonstrate infection of T cells in cord blood from healthy donors. Our results are consistent with transient infection of some T cells being part of normal asymptomatic infection by EBV in young children. IMPORTANCE EBV contributes to several types of human cancer. Some cancers and nonmalignant lymphoproliferative diseases involving T or NK cells contain EBV. These diseases are relatively frequent in Japan and China and have been shown sometimes to have deletions in the EBV genome in the disease cells. We identify further examples of deletions within the EBV genome associated with T or NK cell diseases, and we provide evidence that the virus genomes with these deletions are most likely selected in the individual cases, rather than being transmitted between people during infection. We demonstrate EBV infection of cord blood T cells by highly characterized, cloned EBV genomes and suggest that transient infection of T cells may be part of normal asymptomatic infection by EBV in young children

Identification and characterization of a novel non-structural protein of bluetongue virus

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell

Anterolateral Bone Window for Revision Broken Cemented Stem of Unipolar Hemiarthroplasty

Background. Fractured stem of the hip prosthesis is well documented in the literature. Although it is rare, it is considered as a challenging problem. Many techniques have been described to solve this problem. Purpose of the Study. Evaluation of the effect of anterolateral bone window for extraction of the cemented femoral stem of hemiarthroplasty in revision total hip replacement. Methods. The study included eight revision hip arthroplasties in eight patients, with a broken stem of cemented (Thompson) hemiarthroplasty, which has been revised by the anterolateral proximal femoral window. All cases received cemented cups and cement-in-cement stems, except one case who received cementless long stem. Clinical follow-up of cases by Harries hip score (HHS) and X-ray. Results. Functional improvement of HHS of all cases, with no signs of loosening, after a mean follow-up period of 1.5 years. Conclusion. Extraction of broken stem is a challenging procedure. Many techniques have been described for revision of cases with a fractured stem of hip prosthesis, but we think that the anterolateral femoral bone window is a reproducible technique due to the characteristics of simplicity, short-time procedure, less invasive, not requiring extra instruments, and can be successful for most patients

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells

Correction: Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

[This corrects the article DOI: 10.1371/journal.ppat.1006890.]

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

Letter from John C. Brewer to Emma Davis. The first part discusses mutual acquaintances, and John's wishes that Emma will not forget their memories together. The second part, dated for "Wednesday eve", discusses his and Miss Genie's relationship, what Miss Genie thinks of Emma, and Emma not feeling well

EBNA-LP-null LCLs only establish with a memory B cell-like phenotype.

<p><b>A.</b> Flow cytometry plots show the CD27 (x-axis) and IgD status (y-axis) of LCLs established with different viruses (top labels) in different B cell subsets (labels left) from donor B63. Numbers in the plots show the percentage of cells in each quadrant. Naïve B cell-derived LCLs were all assayed on the same day, 39 days post infection. <b>B & C.</b> Graphs showing percentage of (<b>B</b>) CD27+ve or (<b>C</b>) IgD+ve cells from LCLs (based on data from Fig 5A and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006890#ppat.1006890.s013" target="_blank">S13A Fig</a>) produced from either naïve B cells or total B cells. Data points are shown as either EBNA-LP deficient (black square) or wild-type (orange diamond). Horizontal lines show means for each group.</p