Bovine leukemia viral DNA found on human breast tissue is genetically related to the cattle virus

Bovine leukemia virus (BLV) infection is widespread in cattle and associated with B cell lymphoma. In a previousstudy we demonstrated that bovine leukemia viral DNA was detected in human breast tissues and significantly associated with breast cancer. Our current study aimed to determine whether BLV DNA found in humans and cattle at the same geographical region were genetically related. DNA was extracted from the breast tissue of healthy (n = 32) or cancerous women patients (n = 27) and from the blood (n = 30) of cattle naturally infected with BLV, followed by PCR-amplification and partial nucleotide sequencing of the BLV env gene. We found that the nucleotide sequence identity between BLV env gene fragments obtained from human breast tissue and cattle blood ranged from 97.8 to 99.7% and grouped into genotype 1. Thus, our results further support the hypothesis that this virus might cause a zoonotic infection

Exploring the Impact of Bovine Leukemia Virus Proviral Load on Production, and its Potential Use for Control

The main aim of this dissertation was to evaluate the efficacy of a bovine leukemia virus (BLV) control program by selective removal of high proviral load (HPL) BLV-infected subsets. Six chapters are included. 1) To be acquainted with the current understanding on BLV infection, transmission routes, diagnosis, control, and most importantly, BLV proviral load, a literature review was conducted. This review explored the applicability of BLV proviral load in disease diagnosis, BLV transmission risk assessment, and BLV control. 2) We implemented a cross-sectional study to evaluate the impact of BLV proviral load on milk production of dairy cows. Data obtained from nine dairy herds in Alberta, Canada demonstrated a significant reduction in milk, fat, and protein production of HPL cows when compared with the BLV-negative counterparts. 3) The effectiveness of HPL-cow focused BLV control program in reducing BLV prevalence and seroconversions within the herd was evaluated by conducting a 3-year study among ten dairy herds. The BLV prevalence decreased in four herds whereas the BLV incidence was reduced in nine herds, which supported the notion that removal of HPL cows can offer a feasible and economical option for BLV control. 4) A 1.5-year longitudinal study was designed by enrolling subset of cows from BLV-seropositive (further classified into various proviral load categories) and BLV-seronegative group to monitor the dynamics of various parameters such as BLV proviral load, lymphocyte, white blood cell (WBC) count, antibody titer, CD3+, CD4+, CD8+, CD21+, and WC1+ cell proportions. A relatively stable pattern of BLV proviral load, WBC, CD3+, and CD4+ cell proportion was observed, indicating frequent testing might not be required for these parameters in monitoring BLV infection. 5) A cross-sectional study was conducted to investigate the hematological and immunological impact of BLV infection which suggested a simpler categorization of HPL and LPL as an appropriate approach. Additionally, a lower proviral load cut-off was identified as an accurate threshold for identifying HPL cows. 6) Lastly, all the results and findings were thoroughly discussed, and future directions for using HPL-focused strategies as a potential tool for BLV control and management were elaborated

Factors Involved in Immune Modulation of Bovine Leukemia Virus Infection.

Bovine Leukemia Virus (BLV) is an exogenous type-C retrovirus which can induce an aleukemic (AL) stage, a persistent lymphocytotic (PL) stage, or a lymphosarcomatous (LS) stage in infected animals. After experimental infection with BLV, whole blood and serum samples were obtained from 12 holstein heifers for 103 weeks and used to correlate clinical signs (visual observation and white blood cell counts) with changes in humoral immune response (Agar gel immunodiffusion using envelope gp50 as primary antigen, and ELISA and Western immunoblots using whole BLV as antigen). Monoclonal antibodies against BLV envelope gp50 and gag p24 were produced to confirm reactivity to specific proteins for the various assays. To confirm reactivity of monoclonals and produce large amounts of selected proteins for later studies of BLV, the genes for env, gag, and pX were cloned into eucaryotic expression vectors. After transfection of appropriate plasmids, correct expression of cloned genes was confirmed using monoclonals and indirect immunofluorescence for gp50 and p24 and CAT assay for px. Total lymphocyte counts indicated 6 cattle reached the PL stage during the 103 weeks post infection (PI). Cows progressed through three transient disease stages in the first 3 months PI: transient eosinophilia, lymph node enlargement and lymphocytosis. Of the 6 cattle demonstrating early transient lymph node enlargement, 5/6 were AL. Changes in the humoral immune response to BLV were best detected using Western immunoassays. AL cattle were more likely to have an increased antibody response to the gag protein p24 early PI as compared to PL cattle. PL animals usually reached their highest p24 antibody responses after conversion to the PL stage. Antibody response to env gp50 increased as a function of time PI regardless of disease stage. PL cattle tended to recognize a greater number of the lesser gag proteins, p15, p12 and p10 in the first year PI than AL cattle. All cattle decreased markedly in their reactivity to any of these lesser proteins after 51 weeks PI

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

New insights into Bovine Leukemia Virus (BLV) transcriptional regulation

Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic deltaretrovirus infecting cattle and closely related to human T-cell leukemia viruses I and II (HTLV-I and II). Despite the well-established repression of the 5'LTR-driven viral gene expression, we and others have discovered and characterized two alternative viral promoters[1][2][3][4], allowing a high expression of viral miRNAs[2][3] and antisense viral transcripts[4], potentially contributing to tumor progression and to escape from the host immune system. In addition, our data have suggested a collision phenomenon between the RNAPIII transcribing the miRNA cluster and the RNAPII coming in an antisense orientation from the 3’LTR[1] with, as a result, a stalling of both RNA polymerase complexes. These latter results have indicated that transcriptional interference could be seen as a new mechanism used by BLV to regulate its three transcriptional activities.In this work, we investigated the interplay and self-regulation between the three BLV promoter activities and showed putative critical functions of the transcriptional interference to drive or repress BLV transcriptional activities. In addition, we highlighted the implication of new transcription factors in BLV transcriptional and epigenetic regulations but also in BLV-mediated pathogenesis. Overall in this study, we further investigated new alternative ways used by BLV to regulate its transcriptional and epigenetic status and provided new fundamental insights into BLV transcriptional and epigenetic regulations which could explain the escape from the host immune system and/or the BLV-induced pathogenesis.References[1] B. Van Driessche, A. Rodari, N. Delacourt, S. Fauquenoy, C. Vanhulle, A. Burny, O. Rohr and C. Van Lint, Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome, Sci Rep 6, 2016, 31125-31139[2] N. Rosewick, M. Momont, K. Durkin, H. Takeda, F. Caiment, Y. Cleuter, C. Vernin, F. Mortreux, E. Wattel, A. Burny, M. Georges, and A. Van den Broeke, Deep sequencing reveals abundant noncanonical retroviral microRNAs in B-cell leukemia/lymphoma, Proc Natl Acad Sci USA 110, 2013, 2306–2311.[3] R. P. Kincaid, J. M. Burke, C. S. Sullivan, RNA virus microRNA that mimics a B-cell oncomiR, Proc Natl Acad Sci USA 109, 2012, 3077-3082.[4] K. Durkin, N. Rosewick, M. Artesi, V. Hahaut, P. Griebel, N. Arsic, A. Burny, M. Georges and A. Van den Broeke, Characterization of novel Bovine leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral a microRNAs, Retrovirology 13:33, 2016.info:eu-repo/semantics/nonPublishe