Drosophila melanogaster as a model organism for bluetongue virus replication and tropism.

Bluetongue virus (BTV) is the etiological agent of bluetongue (BT), a hemorrhagic disease of ruminants that can cause high levels of morbidity and mortality. BTV is an arbovirus transmitted between its ruminant hosts by Culicoides biting midges (Diptera: Ceratopogonidae). Recently, Europe has experienced some of the largest BT outbreaks ever recorded, including areas with no known history of the disease, leading to unprecedented economic and animal welfare issues. The current lack of genomic resources and genetic tools for Culicoides restricts any detailed study of the mechanisms involved in the virus-insect interactions. In contrast, the genome of the fruit fly (Drosophila melanogaster) has been successfully sequenced, and it is used extensively as a model of molecular pathways due to the existence of powerful genetic technology. In this study, D. melanogaster is investigated as a model for the replication and tropism of BTV. Using reverse genetics, a modified BTV-1 that expresses the fluorescent mCherry protein fused to the viral nonstructural protein NS3 (BTV-1/NS3mCherry) was generated. We demonstrate that BTV-1/NS3mCherry is not only replication competent as it retains many characteristics of the wild-type virus but also replicates efficiently in D. melanogaster after removal of the bacterial endosymbiont Wolbachia pipientis by antibiotic treatment. Furthermore, confocal microscopy shows that the tissue tropism of BTV-1/NS3mCherry in D. melanogaster resembles that described previously for BTV in Culicoides. Overall, the data presented in this study demonstrate the feasibility of using D. melanogaster as a genetic model to investigate BTV-insect interactions that cannot be otherwise addressed in vector species.

Lung progenitors from lambs can differentiate into specialized alveolar or bronchiolar epithelial cells.

BACKGROUND: Airways progenitors may be involved in embryogenesis and lung repair. The characterization of these important populations may enable development of new therapeutics to treat acute or chronic lung disease. In this study, we aimed to establish the presence of bronchioloalveolar progenitors in ovine lungs and to characterize their potential to differentiate into specialized cells. RESULTS: Lung cells were studied using immunohistochemistry on frozen sections of the lung. Immunocytochemistry and flow cytometry were conducted on ex-vivo derived pulmonary cells. The bronchioloalveolar progenitors were identified by their co-expression of CCSP, SP-C and CD34. A minor population of CD34(pos)/SP-C(pos)/CCSP(pos) cells (0.33% ± 0.31) was present ex vivo in cell suspensions from dissociated lungs. Using CD34 magnetic positive-cell sorting, undifferentiated SP-C(pos)/CCSP(pos) cells were purified (>80%) and maintained in culture. Using synthetic media and various extracellular matrices, SP-C(pos)/CCSP(pos) cells differentiated into either club cells (formerly named Clara cells) or alveolar epithelial type-II cells. Furthermore, these ex vivo and in vitro derived bronchioloalveolar progenitors expressed NANOG, OCT4 and BMI1, specifically described in progenitors or stem cells, and during lung development. CONCLUSIONS: We report for the first time in a large animal the existence of bronchioloalveolar progenitors with dual differentiation potential and the expression of specialized genes. These newly described cell population in sheep could be implicated in regeneration of the lung following lesions or in development of diseases such as cancers.

Un rétrovirus endogène actif et infectieux : le cas gypsy chez Drosophila melanogaster.

Retroviruses are viruses that have the ability to synthesize a DNA copy from their RNA genome and to integrate it in the host genome. Sequencing of eukaryotic genomes has revealed the presence of many of these endogenous retrovirus sequences. The mechanisms by which these sequences colonize the genome are still unknown, and the endogenous retrovirus gypsy of Drosophila melanogaster (D. melanogaster) is a powerful experimental model deciphering this process. Gypsy is expressed in gonadic somatic cells and transferred into the oocyte. This critical step is the first one of the endogenization process. Moreover, gypsy has been shown to possess infectious properties, probably due to its envelope gene that is suspected to have been acquired from baculovirus. So far, gypsy is a unique model to understand endogenization of retroviruses.

In vitro cross-species infections using a caprine arthritis encephalitis lentivirus carrying the GFP marker gene.

A caprine arthritis encephalitis virus (CAEV), carrying the green fluorescent protein (GFP) into the tat region was recently reported [Mselli-Lakhal, L., Guiguen, F., Greenland, T., Mornex, J.F., Chebloune, Y., 2006. Gene transfer system derived from the caprine arthritis-encephalitis lentivirus. J. Virol. Meth. 136, 177-184]. This construct, called pK2EGFPH replicated to titres up to 10(5)IU/ml on infection of caprine cells, and could be concentrated to 10(6)IU/ml by ultracentrifugation. In the present study, the pK2EGFPH construct was characterized better and used in cross-species infection studies. The pK2EGFPH virus could transduce GFP protein expression both to goat synovial membrane cells and to an immortalized goat milk epithelial cell line. The pK2EGFPH infected cells were demonstrated to express both GFP protein and CAEV viral proteins, as demonstrated by radioimmunoprecipitation and multinucleated cell formation. However GFP expression could not be maintained over passages. This vector was used to investigate cross-species infectious potential of CAEV. The bovine cell lines MDBK and GBK were found to be sensitive to infection while the human cell lines Hela, A431 and THP-1 were not. The pK2EGFPH vector should prove useful in studies of CAEV tropism both in vitro and in vivo.

Gene transfer system derived from the caprine arthritis-encephalitis lentivirus.

Lentiviruses are attractive candidates for therapeutic vectors, because of their ability to infect non-dividing target cells. Vectors based on HIV-1 efficiently transfer gene expression to a variety of dividing or quiescent cells, but are subject to reservations on safety grounds. Caprine arthritis encephalitis virus (CAEV) is a lentivirus inducing only minor pathology in its natural host and in related species after cross-species transmission. To test the CAEV potential as vector for gene transfer, a cassette expressing the green fluorescent protein (GFP) under control of a CMV promoter was inserted into the CAEV genome, producing the pK2EGFPH vector. When pseudotyped with vesicular stomatitis virus (VSV)-G envelope protein, this vector allowed efficient transfer of GFP expression in human cells (up to 86% of GFP-expressing cells into the TE671 cell line). Three vectors carrying different parts of the viral gag, pol and env genes were then developed, together with a CAEV packaging system. These vectors allowed delimitation of the minimal CAEV sequences necessary for an improvement of vector production compared to the previously described CAEV-based vectors [Mselli-Lakhal et al., 1998. Defect in RNA transport and packaging are responsible for low transduction efficiency of CAEV-based vectors. Arc. Virol. 143, 681-695]. While our previous vectors were produced in a helper/vector system, the present vectors are produced in a helper/free system. However, these vector titers remain lower than those obtained with other lentiviral vectors carrying equivalent packaging sequences. We discuss on possible reasons of such differences and possible improvements.

Wolbachia influences the maternal transmission of the gypsy endogenous retrovirus in Drosophila melanogaster.

UNLABELLED: 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.

In between: gypsy in Drosophila melanogaster reveals new insights into endogenous retrovirus evolution.

Retroviruses are RNA viruses that are able to synthesize a DNA copy of their genome and insert it into a chromosome of the host cell. Sequencing of different eukaryote genomes has revealed the presence of many such endogenous retroviral sequences. The mechanisms by which these retroviral sequences have colonized the genome are still unknown, and the endogenous retrovirus gypsy of Drosophila melanogaster is a powerful experimental model for deciphering this process in vivo. Gypsy is expressed in a layer of somatic cells, and then transferred into the oocyte by an unknown mechanism. This critical step is the start of the endogenization process. Moreover gypsy has been shown to have infectious properties, probably due to its envelope gene acquired from a baculovirus. Recently we have also shown that gypsy maternal transmission is reduced in the presence of the endosymbiotic bacterium Wolbachia. These studies demonstrate that gypsy is a unique and powerful model for understanding the endogenization of retroviruses.

Small ruminant lentivirus proviral sequences from wild ibexes in contact with domestic goats.

Small ruminant lentiviruses (SRLV) are widespread amongst domesticated goats and sheep worldwide, but have not been clearly identified in wild small ruminants, where they might constitute an animal health risk through contamination from local domesticates. SRLV proviruses from three ibexes from the French Alps are described and sequences from their gag gene and long terminal repeats (LTRs) were compared with sequences from local goats and goat/ibex hybrids. The ibex and hybrid proviruses formed a closely related group with <2 % nucleotide difference. Their LTRs were clearly distinct from those of local goats or reference SRLV sequences; however, their gag sequences resembled those from one local goat and reference sequences from caprine arthritis encephalitis virus rather than visna/maedi virus. One SRLV-positive ibex from a distant site shared similarities with the other ibexes studied in both its gag and LTR sequences, suggesting that a distinct SRLV population could circulate in some wild ibex populations.

Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef.

Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes.

Simian immunodeficiency virus Vpr/Vpx proteins kill bystander noninfected CD4+ T-lymphocytes by induction of apoptosis.

The depletion of CD4+ T-lymphocytes central to the immunodeficiency in acquired immunodeficiency syndrome (AIDS) is largely mediated by apoptosis of both infected and uninfected cells, but the mechanisms involved and the viral proteins responsible are still poorly characterized. It has recently been suggested that, in human and simian immunodeficiency virus (HIV) and SIV, Vpr is a major modulator of apoptosis in infected cells. Recently, we have reported on a chimera of caprine arthritis-encephalitis virus (CAEV) carrying vpr/vpx genes from SIVmac239, which is replication competent in goat macrophages but not in lymphocytes or human cells. Despite infection being restricted to macrophages, inoculation of primary goat peripheral blood mononuclear cells (PBMCs) with this chimera induced apoptosis in the lymphocyte population. In addition, when infected goat synovial membrane (GSM) cells were co-cultured with human CD4+ T lymphocyte SupT1 cell line, these CD4+ T cells showed increased apoptosis. The parental CAEV induced no significant apoptosis in goat PBMC cultures or in co-cultures with human SupT1 lymphocytes. This indicates that SIV Vpr/Vpx proteins indeed mediate apoptosis of T-lymphocytes and, moreover, do so without the need for active infection of these cells. Moreover, this apoptosis was observed when SupT1s were cocultured in direct contact, but not in absence of contact with CAEV-pBSCAvpxvpr-infected GSM cells. In view of these data, we propose that SIV Vpx/Vpr activate cell-to-cell contact-dependent extracellular signaling pathways to promote apoptotic death of uninfected bystander T-lymphocytes. Understanding this mechanism might bring insight for intervening in the loss of CD4+ T lymphocytes in the SIV infection model and in human AIDS.

Clearance of a productive lentivirus infection in calves experimentally inoculated with caprine arthritis-encephalitis virus.

Lentiviruses have long been considered host-specific pathogens, but several recent observations demonstrated their capacity to conquer new hosts from different species, genera, and families. From these cross-species infections emerged new animal and human infectious diseases. The successful colonization and adaptation of a lentivirus to a nonnatural host depends on unspecific and specific host barriers. Some of those barriers exert a relative control of viral replication (i.e., cytotoxic T-lymphocyte response, viral inhibitory factors), but none of them was found able to totally clear the infection once the retrovirus is fully adapted in its host. In this study we examined the evolution of the host-lentivirus interactions occurring in an experimental animal model of cross-species infection in order to analyze the efficiency of those barriers in preventing the establishment of a persistent infection. Five newborn calves were inoculated with caprine arthritis-encephalitis virus (CAEV), and the evolution of infection was studied for more than 12 months. All the animals seroconverted in the first 0.75 to 1 month following the inoculation and remained seropositive for the remaining time of the experiment. Viral infection was productive during 4 months with isolation of replication competent virus from the blood cells and organs of the early euthanized animals. After 4 months of infection, neither replication-competent virus nor virus genome could be detected in blood cells or in the classical target organs, even after an experimental immunosuppression. No evidence of in vitro restriction of CAEV replication was observed in cells from tissues explanted from organs of these calves. These data provide the demonstration of a natural clearance of lentivirus infection following experimental inoculation of a nonnatural host, enabling perspectives of development of new potential vaccine strategies to fight against lentivirus infections.

Specific G2 arrest of caprine cells infected with a caprine arthritis encephalitis virus expressing vpr and vpx genes from simian immunodeficiency virus.

Primate lentivirus (HIV and SIV) vpr accessory genes encode 12- to 14-kDa proteins which induce cell cycle arrest at the G2 phase of infected cells, preventing them from going through mitosis. Members of the HIV-2/SIVmac/SIVsmm group also encode a second closely related accessory protein called Vpx. Vpx and HIV Vpr are critical for virus replication in nondividing cells due to their participation in nuclear import of the preintegration complex. Caprine arthritis encephalitis virus (CAEV) and maedi visna virus are the natural lentiviruses of domestic goat and sheep, respectively, and their genomes do not carry vpr and vpx genes. In this study, we generated chimeric CAEV-based genomes carrying vpr and vpx genes from SIVmac239 and tested their ability to induce G2 cell cycle arrest in infected caprine cells. CAEV-pBSCAvpxvpr is the chimeric genome that was shown to be infectious and replication competent. Our data demonstrated that CAEV-pBSCAvpxvpr-infected goat synovial membrane cell monolayer developed more cytopathic effects and a high proportion of cells remained in the G2 phase of cell cycle. This G2 arrest was observed both at the early and at the late stages of infection, while minimal effect was observed with the parental CAEV-pBSCA. These results, described for the first time in mammalian cells other than those of primates, indicate that Vpr-induced G2 cell cycle arrest is not restricted to only primate cells. Thus, conservation of Vpx/Vpr protein functions in caprine cells suggests a possible role for these proteins in the virus life cycle and its ability to adapt to new hosts. The data presented here thus raise a pertinent question about the biological significance of the conservation of Vpr and Vpx functions in caprine cells despite the high phylogenic distance between primates and small ruminants.