Francois Guiguen

Lack of Functional Receptors Is the Only Barrier That Prevents Caprine Arthritis-Encephalitis Virus from Infecting Human Cells

ABSTRACT Barriers to replication of viruses in potential host cells may occur at several levels. Lack of suitable and functional receptors on the host cell surface, thereby precluding entry of the virus, is a frequent reason for noninfectivity, as long as no alternative way of entry (e.g., pinocytosis, antibody-dependent adsorption) can be exploited by the virus. Other barriers can intervene at later stages of the virus life cycle, with restrictions on transcription of the viral genome, incorrect translation and posttranslational processing of viral proteins, inefficient viral assembly, and release or efficient early induction of apoptosis in the infected cell. The data we present here demonstrate that replication of caprine arthritis-encephalitis virus (CAEV) is restricted in a variety of human cell lines and primary tissue cultures. This barrier was efficiently overcome by transfection of a novel infectious complete-proviral CAEV construct into the same cells. The successful infection of human cells with a vesicular stomatitis virus (VSV) G-pseudotyped Env-defective CAEV confirmed that viral entry is the major obstacle to CAEV infection of human cells. The fully efficient productive infection obtained with the VSV-G-protein-pseudotyped infectious CAEV strengthened the evidence that lack of viral entry is the only practical barrier to CAEV replication in human cells. The virus thus produced retained its original host cell specificity and acquired no propensity to propagate further in human cultures.

Drosophila melanogaster as a Model Organism for Bluetongue Virus Replication and Tropism

ABSTRACT 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.

Clearance of a Productive Lentivirus Infection in Calves Experimentally Inoculated with Caprine Arthritis-Encephalitis Virus

ABSTRACT 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.

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.

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

ABSTRACT 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. 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.