Bacillus thuringiensis serovar israelensis harbours, in addition to several circular plasmids, a small linear molecule of about 15 kb. Sequence analysis of this molecule, named pGIL01, showed the presence of at least 30 ORFs, five of which displayed similarity with proteins involved in phage systems: a B-type family DNA polymerase, a LexA-like repressor, two potential muramidases and a DNA-packaging protein (distantly related to the P9 protein of the tectiviral phage PRD1). Experimental evidence confirmed that pGIL01 indeed corresponds to the linear prophage of a temperate phage. This bacteriophage, named GIL01, produces small turbid plaques and is sensitive to organic solvents, which suggests the presence of lipid components in its capsid. Experiments using proteases and exonucleases also revealed that proteins are linked to the genomes of both pGIL01 prophage and GIL01 phage at their 59 extremities. Altogether, these features are reminiscent of those of phages found in the Tectiviridae family, and more specifically of those of PRD1, a broad-host-range phage of Gram-negative bacteria. Dot-blot hybridization, PFGE, PCR and RFLP analyses also showed the presence of pGIL01 variants in the Bacillus cereus group. INTRODUCTIONBacillus thuringiensis, the most widely used entomopathogenic bacterium, belongs to the Bacillus cereus sensu lato group. This cluster also includes B. cereus sensu stricto, an opportunist organism implicated in food poisoning (Granum & Lund, 1997), Bacillus anthracis, a human and animal pathogen, Bacillus mycoides and Bacillus pseudomycoides, characterized by their rhizoid growth (Nakamura, 1998), and the psychrotolerant Bacillus weihenstephanensis (Lechner et al., 1998). Despite their broad virulence, these bacteria are genetically closely related (their 16S RNA gene sequences share more than 99 % identity) and could be regarded as pertaining to a single species (Ash et al., 1991;Helgason et al., 2000).B. thuringiensis strains produce, during their sporulation, crystal toxins (delta-endotoxins) that are highly toxic to a number of insect larvae belonging to the orders Lepidoptera, Diptera and Coleoptera, but harmless to vertebrates. Classically, the numerous entomopathogenic B. thuringiensis strains, which have their own specific insecticidal activity, have been classified in different serotypes on the basis of their flagellar antigens. B. thuringiensis serovar (sv.) israelensis is active against dipteran species and is therefore one of the bioinsecticides of choice to control black flies and mosquitoes, both important vectors of human and animal diseases (for a recent review, see Glare & O'Callaghan, 2000).B. thuringiensis sv. israelensis strain H14 has been reported to contain at least eight DNA molecules, including three small (5?4, 6?7 and 7?6 kb) and four large (128, 145, 240 and 350 kb) circular plasmids, and one linear molecule (G. Jensen & L. Andrup, unpublished results). The pathogenicity of this strain only depends on the presence of the 128 kb plasmid which encodes the Cry and Cyt...

Lentiviral vectors (LV) are competent gene transfer vehicles, as used for both research and gene therapy applications, because of their stable integration in non-dividing and dividing cells and long-term transgene expression. Along with our understanding that LV offer solutions for gene therapy, biosafety concerns have uncovered risks due to insertional mutagenesis, the generation of replication competent lentiviruses (RCL) and vector mobilization. Researchers therefore continue to devote significant efforts in designing LV with improved efficacy and biosafety features.The choice of a particular LV system for experimental studies is often driven by functional considerations, including increased productivity and/or transduction efficiency. The design of safer vectors has also directly benefited researchers allowing them to conduct experimental studies with lower risk. Currently, vectors combine improved safety features (that decrease the risk of recombination and vector mobilization) with increased transduction efficiency. Hence, risks associated with the inadvertent transduction of cells of the investigator gain greater importance in assessing the overall risk of these vectors and become an important biosafety concern.This review outlines the different strategies used to improve LV biosafety by comparing state-of-the-art and emerging LV production systems and highlighting biosafety issues that can arise during their contained use. The few existing national and international biosafety recommendations that specifically address the use of LV in research are discussed and recommendations for most common research activities using LV are proposed.

The modified vaccinia virus Ankara (MVA) strain is a highly attenuated strain of vaccinia virus that has been demonstrated to be safe for humans. MVA is widely considered as the vaccinia virus strain of choice for clinical investigation because of its high safety profile. It also represents an excellent candidate for use as vector system in recombinant vaccine development for gene delivery or vaccination against infectious diseases or tumours, even in immunocompromised individuals. The use of MVA and recombinant MVA vectors must comply with various regulatory requirements, particularly relating to the assessment of potential risks for human health and the environment. The purpose of the present paper is to highlight some biological characteristics of MVA and MVA-based recombinant vectors and to discuss these from a biosafety point of view in the context of the European regulatory framework for genetically modified organisms with emphasis on the assessment of potential risks associated with environmental release.

One of the most notable characteristics of Tectiviridae resides in their double-layer coats: the doublestranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and NS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating from Bacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated from B. thuringiensis serovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by the Bacillus cereus reference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to the Tectiviridae.The bacteriophage GIL01 has a linear double-stranded DNA genome of 14,931 bp delineated by imperfect 73-bp inverted repeats and protected by proteins at its 5Ј ends. GIL01 is a temperate bacteriophage that was isolated from Bacillus thuringiensis serovar Israelensis whose prophage form, designated pGIL01, resides in the host cell as an autonomous linear plasmid without any apparent integration into the host chromosome. It has been suggested that GIL01 belongs to the family Tectiviridae (28), whose members are characterized by the presence of an internal lipid membrane. While tectiviruses infecting gram-negative bacteria have been extensively studied at the genetic level, those infecting gram-positive bacteria remain less well characterized. PRD1, for instance, is the family model and infects bacteria harboring conjugative plasmids of the P, N, or W incompatibility groups, such as Escherichia coli and Salmonella enterica. These plasmids encode the phage receptor but are not otherwise involved in the virus life cycle In addition to GIL01, three other tectiviruses that infect gram-positive bacteria have been identified so far. The bacteriophages AP50 and NS11, isolated from Bacillus anthracis (19) and Bacillus acidocaldarius (23, 24), respectively, have only been morphologically characterized. Bam35 was isolated from B. thuringiensis serovar Alesti in 1978 (1) and was recently sequenced (21), revealing that it differs from GIL01 by 11 bp. A p...

Bacteriophages are bacterial viruses and consist of a single-or double-stranded DNA or RNA protected by a protein capsid. They are able to infect bacteria by injecting their nucleic acids inside the host. The viruses multiply and induce lysis of the host cell, or they are stabilized as prophage, either inserted in the bacterial genome or as independent plasmid molecules. Bacteriophages represent the most numerous micro-organisms found on earth and play a major role in bacterial evolution by serving as a genomic reservoir in the environment and by promoting lateral gene transfer among bacteria through transduction. They also play a role in bacterial virulence through lysogenic conversion by encoding virulence factors. Bacteriophages, as well as their recombinant derivatives, are now used in a multitude of applications in the biotechnology and medical fields (e.g., as an alternative to antibiotics; tools for screening libraries of proteins, peptides or antibodies; vectors for protein and DNA vaccines; or as gene therapy delivery vehicles). Although most bacteriophages do not represent a threat to human health (unless they are carrying virulence factors), the use of recombinant viral particles in some instances might raise some biosafety concerns by bringing and potentially disseminating new genetic traits among bacterial populations. A thorough risk assessment evaluating the properties of the manipulated bacteriophages may be required to implement adequate containment and control measures to protect human health and the environment. This article describes the general characteristics of bacteriophages that could pose a risk to human health and the environment. Several aspects that should be addressed when manipulating them in laboratories are discussed, with illustrations of relevant examples. Finally, based on the risk assessment conclusion, biosafety recommendations (work practices, safety equipment, and waste management) are proposed.