Population differentiation and Wolbachia phylogeny in mosquitoes of the Aedes scutellaris group

Mosquito species of the Aedes (Stegomyia) scutellaris (Walker) group (Diptera: Culicidae) are distributed across many islands of the South Pacific and include major regional vectors of filariasis, such as Aedes polynesiensis (Marks). Analysis of populations of Ae. polynesiensis at the extremes of its range, from Fiji and from Moorea, French Polynesia, using the rDNA ITS2 (internal transcribed spacer 2) region and six microsatellite markers showed considerable genetic differentiation between them (F(ST) = 0.298-0.357). Phylogenetic analysis of the Wolbachia endosymbionts in three members of the complex revealed that based on the wsp gene they are all very similar and belong to the Mel subgroup of the A clade, closely related to the Wolbachia strain present in the gall wasp Callyrhytis glandium (Giraud) (Hymenoptera: Cynipidae). By contrast they are only distantly related to the A-clade Wolbachia in Aedes albopictus (Skuse), a species closely allied to the Ae. scutellaris group. There was very low differentiation between the Wolbachia in the Moorea and Fiji populations of Ae. polynesiensis.     

Mosquito vectors of the 1998-1999 outbreak of Rift Valley Fever and other arboviruses (Bagaza, Sanar, Wesselsbron and West Nile) in Mauritania and Senegal

Following an outbreak of Rift Valley fever (RVF) in south-eastern Mauritania during 1998, entomological investigations were conducted for 2 years in the affected parts of Senegal and Mauritania, spanning the Sénégal River basin. A total of 92 787 mosquitoes (Diptera: Culicidae), belonging to 10 genera and 41 species, were captured in light traps. In Senegal, Culex poicilipes (41%) and Mansonia uniformis (39%) were the most abundant species caught, whereas Aedes vexans (77%) and Cx. poicilipes (15%) predominated in Mauritania. RVF virus was isolated from 63 pools of Cx. poicilipes: 36 from Senegal in 1998 and 27 from Mauritania in 1999. These results are the first field evidence of Cx. poicilipes naturally infected with RVFV, and the first isolations of this virus from mosquitoes in Mauritania - the main West African epidemic and epizootic area. Additional arbovirus isolates comprised 25 strains of Bagaza (BAG) from Aedes fowleri, Culex neavei and Cx. poicilipes; 67 Sanar (ArD 66707) from Cx. poicilipes; 51 Wesselsbron (WSL) from Ae. vexans and 30 strains of West Nile (WN) from Ma. uniformis, showing differential specific virus-vector associations in the circulation activity of these five arboviruses.     

Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae

Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.     

C- versus N-terminally linked melittin-polyethylenimine conjugates: the site of linkage strongly influences activity of DNA polyplexes

BACKGROUND: One major barrier limiting the transfection efficiency of polyplexes is poor endosomal release, especially when small particles are applied. In an approach to overcome this barrier, covalent attachment of the membrane-active peptide all-(L)-melittin to polyethylenimine (PEI) polyplexes was found to enhance gene transfer efficiency. METHODS: The N-terminus of natural all-(L)- or non-immunogenic all-(D)-melittin was covalently coupled to PEI. In addition, two different all-(D)-melittin conjugates were synthesized, with PEI covalently attached to either the C-terminus (C-mel-PEI) or the N-terminus of melittin (N-mel-PEI). Melittin-PEI polyplexes with particle sizes < 150 nm were generated in HEPES-buffered glucose and tested in transfection experiments. The membrane lytic activities of conjugates and polyplexes were analyzed at neutral and endosomal pH. RESULTS: All-(D)-melittin conjugates mediated enhanced gene expression similar to the natural all-(L)-stereoisomer, with up to 160-fold higher luciferase activity than unmodified PEI. The site of melittin linkage strongly influenced the membrane-destabilizing activities of both conjugates and polyplexes. C-mel-PEI was highly lytic at neutral pH and therefore elevated doses of C-mel-PEI polyplexes induced high toxicity. In contrast, N-mel-PEI was less lytic at neutral pH but retained higher lytic activity than C-mel-PEI at endosomal pH. This apparently promoted better endosomal release of N-mel-PEI polyplexes resulting in efficient gene delivery in different cell lines. CONCLUSIONS: The high potency of C-mel-PEI to destabilize membranes at neutral pH is presumably due to a reported destabilization mechanism proceeding through membrane insertion of the peptide. In contrast, N-mel-PEI is supposed to induce lysis by insertion-independent pore formation according to the toroidal pore model.     

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.     

Viral vectors as tools to model and treat neurodegenerative disorders

The identification of disease-causing genes in familial forms of neurodegenerative disorders and the development of genetic models closely replicating human central nervous system (CNS) pathologies have drastically changed our understanding of the molecular events leading to neuronal cell death. If these achievements open new opportunities of therapeutic interventions, including gene-based therapies, the presence of the blood-brain barrier and the post-mitotic and poor regenerative nature of the target cells constitute important challenges. Efficient delivery systems taking into account the specificity of the CNS are required to administer potential therapeutic candidates. In addition, genetic models in large animals that replicate the late stages of the diseases are in most cases not available for pre-clinical studies. The present review summarizes the potential of viral vectors as tools to create new genetic models of CNS disorders in various species including primates and the recent progress toward viral gene therapy clinical trials for the administration of therapeutic candidates into the brain.     

Characterization of antiproliferative and cytotoxic properties of the HSV-1 immediate-early ICPo protein

BACKGROUND: The identification of novel proteins displaying cytostatic and/or cytotoxic actions that could eventually be used for gene therapy is a major issue in cancer research. Data from the literature suggested that the immediate-early ICP0 protein from herpes simplex virus type 1 (HSV-1) could fulfill these properties as it had been observed that this protein is involved in arrest of cell growth at the G1/S and G2/M phases of the cell cycle and that deletion of ICP0 from HSV-1 or HSV-1-recombinant vectors significantly reduced their cytotoxicity. The molecular basis of its action is likely related to the ability of ICP0, which possesses E3-ubiquitin ligase activity, to target destruction of key cellular proteins, including centromeric proteins, resulting in abnormal chromosome segregation, unusual cytokinesis, and emergence of nuclear morphological aberrations. However, neither the gene therapy potential of ICP0 on its own nor its action on primary quiescent cells has been assessed to date. The aim of this work was therefore to evaluate the antiproliferative and cytotoxic properties of ICP0 on a human glioblastoma cell line and on quiescent primary cells, and to explore whether this protein has a potential for gene therapy of cancer. METHODS: HSV-1-based amplicon particles were generated following a recently described method that produces relatively high titers of vector stocks that are essentially free of helper virus. These vectors express either wild-type ICP0 or FXE, a RING finger mutated inactive form of ICP0, together with reporter green fluorescent protein (GFP). The vectors were used to infect Gli36 cells, which derive from a human glioblastome, and cultured rat primary cardiomyocytes and brain cells, two well-established models of non-dividing cells. Expression and localization of ICP0 and FXE, as well as their action on centromeres and nuclear morphology, were evaluated by Western blotting, indirect immune fluorescence, and confocal microscopy using specific antibodies and DAPI labeling. The impact of ICP0 on cell growth, toxicity, and viability was evaluated in the different cells using a variety of methods, including FACS analysis after propidium iodide and AnnexinV staining, crystal violet staining, clonogenic capability, caspase 3 activation, MTT tests, and release of lactate dehydrogenase, after infection with the different vectors. RESULTS: The three cell types under study showed high levels of transduction by amplicons and strong expression of GFP, ICP0, and FXE transgenic proteins. Wild-type ICP0, but not FXE, induced centromeric disruption, appearance of micronuclei, arrest of proliferation, and significant cell death in glioblastoma Gli36 cells. In contrast, neither micronuclei formation nor any other sign of cell toxicity could be observed in cultured primary cardiomyocytes or brain cells, as evaluated by MTT tests and crystal violet staining. Furthermore, in the case of cardiomyocytes, expression of ICP0 did not interfere with beating as cells continued to beat at the same frequency as non-infected cells for several days post-infection. Neither AnnexinV early staining nor caspase 3 activation was observed in dying infected Gli36 cells, suggesting that these cells were not entering apoptosis. In contrast, release of lactate dehydrogenase by infected Gli36 cells suggested a necrotic way of death. CONCLUSIONS: ICP0 induced a strong cytostatic action followed by significant cell death on the glioblastoma Gli36 cell line. In contrast, neither cell death nor any other evidence of ICP0-induced toxicity affecting major physiological parameters was observed in primary cultured cardiomyoctes and brain cells, two models of primary non-cycling cells. These results suggest that ICP0 has gene therapy potential and could represent the first member of a novel family of directly acting proteins that could be used to treat cancers.