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.

Prevalence of West Nile virus neutralizing antibodies in wild birds from the Camargue area, southern France.

The Camargue area of southern France experienced the re-emergence of West Nile Virus (WNV) in the late summer of 2000 and 2004. Immediately preceding the 2004 outbreak, samples were collected from 432 birds of 32 different species captured in mist nets and from 201 Cattle Egret (Bubulcus ibis) nestlings sampled in their nests between 1 April and 12 June 2004. West Nile virus neutralizing titers of >/=40 were detected in 4.8% (95% confidence limit, 2.9-7.5%) of the adult birds and in 1.6% (0.3-4.6%) of the egret nestlings. Migratory passerines had a higher prevalence of WNV neutralizing antibodies (7.0%) than did resident and short-distance migratory passerines (0.8%), suggesting exposure to WNV or a related flavivirus during overwintering in Africa.

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.

Magpies as hosts for West Nile virus, southern France.

European magpies (Pica pica) from southern France were tested for antibodies to West Nile virus (WNV) and viral shedding in feces during spring-autumn 2005. Results suggest that this peridomestic species may be a suitable sentinel species and a relevant target for additional investigations on WNV ecology in Europe.

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.

Jaagsiekte Sheep Retrovirus (JSRV): from virus to lung cancer in sheep.

Jaagsiekte Sheep Retrovirus (JSRV) is a betaretrovirus infecting sheep. This virus is responsible for a pulmonary adenocarcinoma, by transformation of epithelial cells from the bronchioli and alveoli. This animal cancer is similar to human bronchioloalveolar cancer (BAC), a specific form of human lung cancer for which a viral aetiology has not yet been identified. JSRV interacts with target cells through the membrane receptor Hyal2. The JSRV genome is simple and contains no recognised oncogene. It is now well established that the viral envelope protein is oncogenic by itself, via the cytoplasmic domain of the transmembrane glycoprotein and some domains of the surface glycoprotein. Activation of the PI3K/Akt and MAPK pathways participates in the envelope-induced transformation. Tumour development is associated with telomerase activation. This review will focus on the induction of cancer by JSRV.

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.

Mammary transmission of caprine arthritis encephalitis virus: a 3D model for in vitro study.

Transmission of Caprine Arthritis Encephalitis virus (CAEV) from the mother to offspring is principally mediated by infected cells from colostrum and milk. The infection of the dam is often sub-clinical, and results in increased cellularity of milk, sometimes exacerbated by bacterial co-infections. Although monocytes are the major viral host cells, several other cell types, including epithelial mammary cells, fibroblasts and endothelial cells show low levels of in vivo infection. In vitro, however, all phenotypes of mammary gland cells are individually highly sensitive to CAEV infection. This suggests that local mechanisms act to control viral expression. Our goal is to analyse the mechanisms regulating local virus infection, including the physiological status of the mammary gland and bacterial co-infections. In this work, we present the development of a model for the in vitro reconstitution of mammary gland tissue using 3D cultures in Matrigel. Mononuclear cells from the blood are added to the 3D cultures in vitro. In these experimental conditions, the mammary cells spontaneously organize into mammospheres. Blood leucocytes migrate into the culture gel, and localize particularly at the periphery of the mammospheres. Mammospheres were susceptible to infection in vitro by CAEV, as shown by a cytopathic effect and expression of late CAEV antigen p30. This model will allow the in vitro study of virus expression, transfer of infection to mammary gland cells and interactions between the mammary gland cells, infected monocytes and immunocompetent cells. It will allow the study of mechanisms participating in the control of passage of pathogens into milk, according to the physiological and CAEV-infection status of the animal, microenvironment and the presence of bacterial co-infections.

Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: worldwide emergence.

Infections caused by community-acquired (CA)-methicillin--resistant Staphylococcus aureus (MRSA) have been reported worldwide. We assessed whether any common genetic markers existed among 117 CA-MRSA isolates from the United States, France, Switzerland, Australia, New Zealand, and Western Samoa by performing polymerase chain reaction for 24 virulence factors and the methicillin-resistance determinant. The genetic background of the strain was analyzed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The CA-MRSA strains shared a type IV SCCmec cassette and the Panton-Valentine leukocidin locus, whereas the distribution of the other toxin genes was quite specific to the strains from each continent. PFGE and MLST analysis indicated distinct genetic backgrounds associated with each geographic origin, although predominantly restricted to the agr3 background. Within each continent, the genetic background of CA-MRSA strains did not correspond to that of the hospital-acquired MRSA.

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.

Cross-species reactivity of seven monoclonal antibodies with equine lymphocytes by flow cytometry.

The recognition of equine lymphocyte antigens by monoclonal antibodies (mAbs) directed against human CD11a, CD18, CD21, CD23, CD29 and DR, as well as mouse CD23 was studied by flow cytometry. Unlike anti-CD11a, -CD21, -CD23 and DR mAbs, anti-CD18 and CD29 mAbs labelled the same percentage of horse peripheral blood lymphocytes (PBL) as human PBL. Double-staining with anti-horse immunoglobulin antibodies showed that anti-CD21 and -CD23 mAbs are mainly bound to peripheral blood B lymphocytes. The seven mAbs were also tested on the lymph node and thymus cells. The molecular targets of anti-CD11a, CD18 and CD29 mAbs were confirmed by immunoprecipitation of the membrane proteins. Our results suggest that anti-CD18, -CD29 and -DR mAbs recognise similarly expressed molecular homologues on equine cells, but that anti-CD11a, -CD21 and -CD23 mAbs recognise either different molecules or homologues that are expressed at different levels on horse cells.

High genetic variability of the agr locus in Staphylococcus species.

The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.

Aseptic meningitis after neurosurgery: a demonstration of bacterial involvement.

OBJECTIVE: To evaluate the presence of bacteria in samples from patients suffering from 'aseptic' meningitis following craniotomy. METHODS: Prospective study in which cerebrospinal fluid (CSF) from patients suffering from post-craniotomy meningitis and negative control patients were submitted to conventional culture and to polymerase chain reaction (PCR) using bacterial 16S rRNA universal primers, followed in some cases by DNA sequencing of the PCR product and phylogenetic analysis. RESULTS: CSF from patients with either culture-positive or culture-negative meningitis yielded positive amplifications, whereas no amplification was obtained with CSF from control patients. All positive signals were confirmed by Southern hybridization with a prokaryote 16S RNA-specific probe. Six PCR products, of which three were collected from later cases of culture-negative meningitis, were cloned and sequenced. Sequence analysis suggested affinities with Pseudomonas in three cases, with Escherichia in two cases and with Rhodococcus in one case. CONCLUSIONS: Many cases of culture-negative (aseptic) meningitis are probably bacterial meningitis and justify antibiotic treatment. The bacteria responsible for these cases of culture-negative meningitis might have peculiar growth requirements in vitro.