Franchissement de la barrière d’espèces du virus de l’arthrite et de l’encéphalite de la chèvre (CAEV) des ongulés domestiques vers les sauvages : analyse des propriétés biologiques et génétiques.

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Wolbachia Influences the Maternal Transmission of the gypsy Endogenous Retrovirus in Drosophila melanogaster

The endosymbiotic bacteria of the genus Wolbachia are present in most insects and are maternally transmitted through the germline. Moreover, these intracellular bacteria exert antiviral activity against insect RNA viruses, as in Drosophila melanogaster, which could explain the prevalence of Wolbachia bacteria in natural populations. Wolbachia is maternally transmitted in D. melanogaster through a mechanism that involves distribution at the posterior pole of mature oocytes and then incorporation into the pole cells of the embryos. In parallel, maternal transmission of several endogenous retroviruses is well documented in D. melanogaster. Notably, gypsy retrovirus is expressed in permissive follicle cells and transferred to the oocyte and then to the offspring by integrating into their genomes. Here, we show that the presence of Wolbachia wMel reduces the rate of gypsy insertion into the ovo gene. However, the presence of Wolbachia does not modify the expression levels of gypsy RNA and envelope glycoprotein from either permissive or restrictive ovaries. Moreover, Wolbachia affects the pattern of distribution of the retroviral particles and the gypsy envelope protein in permissive follicle cells. Altogether, our results enlarge the knowledge of the antiviral activity of Wolbachia to include reducing the maternal transmission of endogenous retroviruses in D. melanogaster. IMPORTANCE Animals have established complex relationships with bacteria and viruses that spread horizontally among individuals or are vertically transmitted, i.e., from parents to offspring. It is well established that members of the genus Wolbachia, maternally inherited symbiotic bacteria present mainly in arthropods, reduce the replication of several RNA viruses transmitted horizontally. Here, we demonstrate for the first time that Wolbachia diminishes the maternal transmission of gypsy, an endogenous retrovirus in Drosophila melanogaster. We hypothesize that gypsy cannot efficiently integrate into the germ cells of offspring during embryonic development in the presence of Wolbachia because both are competitors for localization to the posterior pole of the egg. More generally, it would be of interest to analyze the influence of Wolbachia on vertically transmitted exogenous viruses, such as some arboviruses

Azole fungicides in zebrafish: new effects for old molecules

Azole is a class of diverse compounds discovered several decades ago and essentially used as antifungals in agriculture and medicine. Their primary mode of action is to inhibit the fungal enzyme 14α-demethylase, which produces ergosterol, an important component of the cell membranes of fungi. Despite this specific mode of action, azoles are also characterized by their capacity to disrupt the endocrine system of vertebrate through multiple mechanisms notably by altering steroidogenesis, a key physiological process responsible for the biosynthesis of steroidal hormones. For instance, azole compounds affect both expression and enzymatic activities of several steroidogenic enzymes in vertebrate models, including fish, leading to reproductive disorders. Because of their uses, their presence in the aquatic environment (surface waters of rivers, lakes and estuaries; sewage sludge) has been recently reported in different industrialized countries raising the need to assess hazard and risk posed to aquatic organisms. In this context, several experiments have been performed to explore the effects of the pharmaceutical azole, clotrimazole, on the endocrine system in the zebrafish. In males, we found that clotrimazole was able to affect the testicular physiology by affecting steroidogenesis, androgen release and spermatogenesis (Hinfray et al., 2011, Baudiffier et al. 2012, 2013). However, the most striking effect was observed in females. Indeed, we found that exposure of adult female zebrafish to clotrimazole led to a dramatic masculinisation as revealed by the complete sex-reversal of the phenotypic sex. Remarkably, this sex-reversal occurred rapidly leading to well-differentiated testicular tissue after 42 days of exposure. By using cyp19a1a-GFP transgenic zebrafish, we further demonstrated that clotrimazole led to a time-dependent inhibition of GFP expression in ovary which preceded the histological differentiation of testis demonstrating the crucial role played by aromatase in the process of masculinisation. Altogether, our study demonstrates that clotrimazole significantly affect the gonad endocrinology and physiology of fish revealing new and striking effects on its ability to reverse the phenotypic sex of adult female. Based on our data, it is clear that further studies are needed to address the issue raised by the presence of azoles in the aquatic environment as regards to their potential impact on wild population of fish

Azole fungicides in zebrafish: new effects for old molecules

International audienceAzole is a class of diverse compounds discovered several decades ago and essentially used as antifungals in agriculture and medicine. Their primary mode of action is to inhibit the fungal enzyme 14α-demethylase, which produces ergosterol, an important component of the cell membranes of fungi. Despite this specific mode of action, azoles are also characterized by their capacity to disrupt the endocrine system of vertebrate through multiple mechanisms notably by altering steroidogenesis, a key physiological process responsible for the biosynthesis of steroidal hormones. For instance, azole compounds affect both expression and enzymatic activities of several steroidogenic enzymes in vertebrate models, including fish, leading to reproductive disorders. Because of their uses, their presence in the aquatic environment (surface waters of rivers, lakes and estuaries; sewage sludge) has been recently reported in different industrialized countries raising the need to assess hazard and risk posed to aquatic organisms. In this context, several experiments have been performed to explore the effects of the pharmaceutical azole, clotrimazole, on the endocrine system in the zebrafish. In males, we found that clotrimazole was able to affect the testicular physiology by affecting steroidogenesis, androgen release and spermatogenesis (Hinfray et al., 2011, Baudiffier et al. 2012, 2013). However, the most striking effect was observed in females. Indeed, we found that exposure of adult female zebrafish to clotrimazole led to a dramatic masculinisation as revealed by the complete sex-reversal of the phenotypic sex. Remarkably, this sex-reversal occurred rapidly leading to well-differentiated testicular tissue after 42 days of exposure. By using cyp19a1a-GFP transgenic zebrafish, we further demonstrated that clotrimazole led to a time-dependent inhibition of GFP expression in ovary which preceded the histological differentiation of testis demonstrating the crucial role played by aromatase in the process of masculinisation. Altogether, our study demonstrates that clotrimazole significantly affect the gonad endocrinology and physiology of fish revealing new and striking effects on its ability to reverse the phenotypic sex of adult female. Based on our data, it is clear that further studies are needed to address the issue raised by the presence of azoles in the aquatic environment as regards to their potential impact on wild population of fish

Expression and localization of steroidogenic enzymes during gonadal sexual differentiation in zebrafish

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Immortalized goat milk epithelial cell lines replicate CAEV at high level

Primary milk epithelial cells were isolated from CAEV-uninfected goats and three cell lines designated TIGMEC-1, TIGMEC-2 and TIGMEC-3 were established. The three cell lines retained their morphological characteristics of epithelial cells and expressed specific epithelial cytokeratin marker as well as the immortalizing SV40 large T antigen. The kinetics of growth of TIGMEC1, TIGMEC2 and TIGMEC3 cell lines showed a doubling time of 24-48 hours while the parental cell lines became senescent after the passage 6 in cell culture. Like the parental primary cells, the three cell lines were found to be highly sensitive to CAEV-pBSCA, an infectious molecular clone of CAEV-CO strain, and to a French isolate CAEV-3112. TIGMEC cell lines infected with CAEV-pBSCA became chronically infected producing high virus titers in absence of cytopathic effects. These cell lines may be useful for study of the possible physiological alterations in mammary epithelial cells infected with small ruminant lentiviruses and their consequences on milk quality. On an other hand, these cell lines can be used to study their role in virus transmission and pathogenesis.Des cellules épithéliales du lait immortalisées répliquent le CAEV avec une grande efficacité. Des cellules épithéliales primaires isolées à partir de chèvres non infectées par le CAEV ont été immortalisées pour dériver 3 lignées appelées TIGMEC-1, TIGMEC-2 et TIGMEC-3. Les cellules des 3 lignées ont conservé leurs caractéristiques morphologiques de cellules épithéliales et expriment la cytokératine, un marqueur spécifique des cellules épithéliales et l'antigène immortalisant T du virus SV40. La courbe de croissance des lignées TIGMEC-1, TIGMEC-2 et TIGMEC-3 montre une multiplication des cellules toutes les 24 à 48 heures alors que les cellules parentales ne se divisent plus après 6 passages en culture. Comme les cellules primaires dont elles dérivent, ces lignées se sont avérées sensibles à l'infection par le CAEV-pBSCA, un clone moléculaire infectieux de la souche CAEV-CO, et un isolat français le CAEV-3112. Les lignées TIGMEC infectées avec le CAEV-pBSCA deviennent chroniquement infectées et produisent des titres élevés de virus en l'absence d'effet cytopathogène. Ces lignées pourront être très utiles pour étudier les altérations physiologiques éventuelles liées à l'infection de cellules épithéliales par les lentivirus des petits ruminants et pouvant avoir des conséquences sur la qualité du lait. Par ailleurs, ces cellules pourraient être utilisées pour étudier le rôle des cellules épithéliales du lait dans la transmission du virus et l'induction de pathologie

Pneumopathies interstitielles diffuses dues aux lentivirus chez l'homme (HIV-1) et chez l'animal

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