Michal Reichert

Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human

In 1871, the observation of yellowish nodules in the enlarged spleen of a cow was considered to be the first reported case of bovine leukemia. The etiological agent of this lymphoproliferative disease, bovine leukemia virus (BLV), belongs to the deltaretrovirus genus which also includes the related human T-lymphotropic virus type 1 (HTLV-1). This review summarizes current knowledge of this viral system, which is important as a model for leukemogenesis. Recently, the BLV model has also cast light onto novel prospects for therapies of HTLV induced diseases, for which no satisfactory treatment exists so far.

Cell dynamics and immune response to BLV infection: a unifying model

Bovine Leukemia virus (BLV) is the natural etiological agent of a lymphoproliferative disease in cattle. BLV can also be transmitted experimentally to a related ruminant species, sheep, in which the pathogenesis is more acute. Although both susceptible species develop a strong anti-viral immune response, the virus persists indefinitely throughout life, apparently at a transcriptionally silent stage, at least in a proportion of infected cells. Soon after infection, these humoral and cytotoxic activities very efficiently abolish the viral replicative cycle, permitting only mitotic expansion of provirus-carrying cells. Short term cultures of these infected cells initially indicated that viral expression protects against spontaneous apoptosis, suggesting that leukemia is a process of accumulation of long-lived cells. This conclusion was recently reconsidered following in vivo dynamic studies based on perfusions of nucleoside (bromodeoxyuridine) or fluorescent protein markers (CFSE). In sheep, the turnover rate of infected cells is increased, suggesting that a permanent clearance process is exerted by the immune system. Lymphocyte trafficking from and to the secondary lymphoid organs is a key component in the maintenance of cell homeostasis. The net outcome of the immune selective pressure is that only cells in which the virus is transcriptionally silenced survive and accumulate, ultimately leading to lymphocytosis. Activation of viral and/or cellular expression in this silent reservoir with deacetylase inhibitors causes the collapse of the proviral loads. In other words, modulation of viral expression appears to be curative in lymphocytic sheep, an approach that might also be efficient in patients infected with the related Human T-lymphotropic virus type 1. In summary, a dynamic interplay between BLV and the host immune response modulates a complex equilibrium between (i) viral expression driving (or) favoring proliferation and (ii) viral silencing preventing apoptosis. As conclusion, we propose a hypothetical model unifying all these mechanisms.

Differentiation between Cyprinid herpesvirus type-3 lineages using duplex PCR

To date, all the isolates of Cyprinid herpesvirus type-3 (CyHV3) responsible for serious outbreaks in carps Cyprinus carpio have been found to be very similar or identical on the basis of DNA sequences of a few reference genes. However, two genetic lineages (U/I and J) are distinguished by full-length genome sequencing. Two molecular markers presenting genetic variations were targeted for developing a duplex PCR assay able to distinguish CyHV3-U/I from CyHV3-J while avoiding DNA sequencing. The method was validated on a series of 42 samples of infected carps from France, The Netherlands and Poland collected from 2001 to 2008. Among these samples, both the U/I and J genotypes were identified, but also a third genotype representing a genetic intermediate between U/I and J for one of the two molecular markers. A classification of CyHV3 genotypes, based on the alleles of the two molecular markers, is proposed. The assay is easy to perform and provides a genotype information with samples moderately or highly concentrated. This tool should improve our knowledge regarding the present distribution and future diversification of this emerging virus.

Inhibition of Histone Deacetylases Induces Bovine Leukemia Virus Expression In Vitro and In Vivo

ABSTRACT Packaging into nucleosomes results in a global transcriptional repression as a consequence of exclusion of sequence-specific factors. This inhibition can be relieved by using inhibitors of histone deacetylases, acetylation being a major characteristic of transcriptionally active chromatin. Paradoxically, the expression of only ∼2% of the total cellular genes is modulated by histone hyperacetylation. To unravel the potential role of this transcriptional control on BLV expression, we tested the effect of two highly specific inhibitors of deacetylases, trichostatin A (TSA) and trapoxin (TPX). Our results demonstrate that treatment with TSA efficiently enhanced long terminal repeat-directed gene expression of integrated reporter constructs in heterologous D17 stable cell lines. To further examine the biological relevance of these observations made in vitro, we analyzed ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected sheep. TSA deacetylase inhibitor induced a drastic increase in viral expression at levels comparable to those induced by treatment with phorbol-12-myristate 13-acetate and ionomycin, the most efficient activators of BLV expression known to date. TSA acted directly on BLV-infected B lymphocytes to increase viral expression and does not seem to require T-cell cooperation. Inhibition of deacetylation after treatment with TSA or TPX also significantly increased viral expression in PBMCs from cattle, the natural host for BLV. Together, our results show that BLV gene expression is, like that of a very small fraction of cellular genes, also regulated by deacetylation.

Reduced Cell Turnover in Bovine Leukemia Virus-Infected, Persistently Lymphocytotic Cattle

ABSTRACT Although nucleotide analogs like bromodeoxyuridine have been extensively used to estimate cell proliferation in vivo, precise dynamic parameters are scarce essentially because of the lack of adequate mathematical models. Besides recent developments on T cell dynamics, the turnover rates of B lymphocytes are largely unknown particularly in the context of a virally induced pathological disorder. Here, we aim to resolve this issue by determining the rates of cell proliferation and death during the chronic stage of the bovine leukemia virus (BLV) infection, called bovine persistent lymphocytosis (PL). Our methodology is based on direct intravenous injection of bromodeoxyuridine in association with subsequent flow cytometry. By this in vivo approach, we show that the death rate of PL B lymphocytes is significantly reduced (average death rate, 0.057 day−1 versus 0.156 day−1 in the asymptomatic controls). Concomitantly, proliferation of the PL cells is also significantly restricted compared to the controls (average proliferation rate, 0.0046 day−1 versus 0.0085 day−1). We conclude that bovine PL is characterized by a decreased cell turnover resulting both from a reduction of cell death and an overall impairment of proliferation. The cell dynamic parameters differ from those measured in sheep, an experimental model for BLV infection. Finally, cells expressing p24 major capsid protein ex vivo were not BrdU positive, suggesting an immune selection against proliferating virus-positive lymphocytes. Based on a comparative leukemia approach, these observations might help to understand cell dynamics during other lymphoproliferative disease such as chronic lymphocytic leukemia or human T-cell lymphotropic virus-induced adult T-cell leukemia in humans.

Fate of premalignant clones during the asymptomatic phase preceding lymphoid malignancy.

Almost all cancers are preceded by a prolonged period of clinical latency during which a combination of cellular events helps move carcinogen-exposed cells towards a malignant phenotype. Hitherto, investigating the fate of premalignant cells in vivo remained strongly hampered by the fact that these cells are usually indistinguishable from their normal counterparts. Here, for the first time, we have designed a strategy able to reconstitute the replicative history of the bona fide premalignant clone in an animal model, the sheep experimentally infected with the lymphotropic bovine leukemia virus. We have shown that premalignant clones are early and clearly distinguished from other virus-exposed cells on the basis of their degree of clonal expansion and genetic instability. Detectable as early as 0.5 month after the beginning of virus exposure, premalignant cells displayed a two-step pattern of extensive clonal expansion together with a mutation load approximately 6 times higher than that of other virus-exposed cells that remained untransformed during the life span of investigated animals. There was no fixation of somatic mutations over time, suggesting that they regularly lead to cellular death, partly contributing to maintain a normal lymphocyte count during the prolonged premalignant stage. This equilibrium was finally broken after a period of 18.5 to 60 months of clinical latency, when a dramatic decrease in the genetic instability of premalignant cells coincided with a rapid increase in lymphocyte count and lymphoma onset.

Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep.

ABSTRACT Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.

Bovine Leukemia Virus Small Noncoding RNAs Are Functional Elements That Regulate Replication and Contribute to Oncogenesis In Vivo

Retroviruses are not expected to encode miRNAs because of the potential problem of self-cleavage of their genomic RNAs. This assumption has recently been challenged by experiments showing that bovine leukemia virus (BLV) encodes miRNAs from intragenomic Pol III promoters. The BLV miRNAs are abundantly expressed in B-cell tumors in the absence of significant levels of genomic and subgenomic viral RNAs. Using deep RNA sequencing and functional reporter assays, we show that miRNAs mediate the expression of genes involved in cell signaling, cancer and immunity. We further demonstrate that BLV miRNAs are essential to induce B-cell tumors in an experimental model and to promote efficient viral replication in the natural host.

Recent advances in BLV research

Different animal models have been proposed to investigate the mechanisms of Human T-lymphotropic Virus (HTLV)-induced pathogenesis: rats, transgenic and NOD-SCID/γcnull (NOG) mice, rabbits, squirrel monkeys, baboons and macaques. These systems indeed provide useful information but have intrinsic limitations such as lack of disease relevance, species specificity or inadequate immune response. Another strategy based on a comparative virology approach is to characterize a related pathogen and to speculate on possible shared mechanisms. In this perspective, bovine leukemia virus (BLV), another member of the deltaretrovirus genus, is evolutionary related to HTLV-1. BLV induces lymphoproliferative disorders in ruminants providing useful information on the mechanisms of viral persistence, genetic determinants of pathogenesis and potential novel therapies.

Reduced levels of reactive oxygen species correlate with inhibition of apoptosis, rise in thioredoxin expression and increased bovine leukemia virus proviral loads

BackgroundBovine Leukemia virus (BLV) is a deltaretrovirus that induces lymphoproliferation and leukemia in ruminants. In ex vivo cultures of B lymphocytes isolated from BLV-infected sheep show that spontaneous apoptosis is reduced. Here, we investigated the involvement of reactive oxygen species (ROS) in this process.ResultsWe demonstrate that (i) the levels of ROS and a major product of oxidative stress (8-OHdG) are reduced, while the thioredoxin antioxidant protein is highly expressed in BLV-infected B lymphocytes, (ii) induction of ROS by valproate (VPA) is pro-apoptotic, (iii) inversely, the scavenging of ROS with N-acetylcysteine inhibits apoptosis, and finally (iv) the levels of ROS inversely correlate with the proviral loads.ConclusionTogether, these observations underline the importance of ROS in the mechanisms of inhibition of apoptosis linked to BLV infection.