Genome Characteristics of a Novel Phage from Bacillus thuringiensis Showing High Similarity with Phage from Bacillus cereus

Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the “late” region, the “lysogeny-lysis” region and the “early” region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies

Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex

<p>Abstract</p> <p>Background</p> <p>The <it>Burkholderia cepacia </it>complex (BCC) is comprised of at least seventeen Gram-negative species that cause infections in cystic fibrosis patients. Because BCC bacteria are broadly antibiotic resistant, phage therapy is currently being investigated as a possible alternative treatment for these infections. The purpose of our study was to sequence and characterize three novel BCC-specific phages: KS5 (vB_BceM-KS5 or vB_BmuZ-ATCC 17616), KS14 (vB_BceM-KS14) and KL3 (vB_BamM-KL3 or vB_BceZ-CEP511).</p> <p>Results</p> <p>KS5, KS14 and KL3 are myoviruses with the A1 morphotype. The genomes of these phages are between 32317 and 40555 base pairs in length and are predicted to encode between 44 and 52 proteins. These phages have over 50% of their proteins in common with enterobacteria phage P2 and so can be classified as members of the <it>Peduovirinae </it>subfamily and the "P2-like viruses" genus. The BCC phage proteins similar to those encoded by P2 are predominantly structural components involved in virion morphogenesis. As prophages, KS5 and KL3 integrate into an AMP nucleosidase gene and a threonine tRNA gene, respectively. Unlike other P2-like viruses, the KS14 prophage is maintained as a plasmid. The P2 <it>E+E' </it>translational frameshift site is conserved among these three phages and so they are predicted to use frameshifting for expression of two of their tail proteins. The <it>lysBC </it>genes of KS14 and KL3 are similar to those of P2, but in KS5 the organization of these genes suggests that they may have been acquired via horizontal transfer from a phage similar to λ. KS5 contains two sequence elements that are unique among these three phages: an IS<it>Bmu</it>2-like insertion sequence and a reverse transcriptase gene. KL3 encodes an EcoRII-C endonuclease/methylase pair and Vsr endonuclease that are predicted to function during the lytic cycle to cleave non-self DNA, protect the phage genome and repair methylation-induced mutations.</p> <p>Conclusions</p> <p>KS5, KS14 and KL3 are the first BCC-specific phages to be identified as P2-like. As KS14 has previously been shown to be active against <it>Burkholderia cenocepacia in vivo</it>, genomic characterization of these phages is a crucial first step in the development of these and similar phages for clinical use against the BCC.</p

The Secret Life of the Anthrax Agent Bacillus anthracis: Bacteriophage-Mediated Ecological Adaptations

Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities

Contained use of Bacteriophages: Risk assessment and biosafety recommendations2463

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 therapydelivery 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.</p

Contained use of bacteriophages: Risk assessment and biosafety recommendations

Bacteriophages are bacterial viruses and consist ofa single- or double-stranded DNA or RNA protected bya protein capsid. They are able to infect bacteria byinjecting their nucleic acids inside the host. The virusesmultiply and induce lysis of the host cell, or they arestabilized as prophage, either inserted in the bacterialgenome or as independent plasmid molecules. Bacteriophagesrepresent the most numerous micro-organismsfound on earth and play a major role in bacterial evolutionby serving as a genomic reservoir in the environmentand by promoting lateral gene transfer amongbacteria through transduction. They also play a role inbacterial virulence through lysogenic conversion byencoding virulence factors. Bacteriophages, as well astheir recombinant derivatives, are now used in a multitudeof applications in the biotechnology and medicalfields (e.g., as an alternative to antibiotics; tools forscreening libraries of proteins, peptides or antibodies;vectors for protein and DNA vaccines; or as gene therapydelivery vehicles). Although most bacteriophagesdo not represent a threat to human health (unless theyare carrying virulence factors), the use of recombinantviral particles in some instances might raise some biosafetyconcerns by bringing and potentially disseminatingnew genetic traits among bacterial populations. Athorough risk assessment evaluating the properties ofthe manipulated bacteriophages may be required toimplement adequate containment and control measuresto protect human health and the environment. This articledescribes the general characteristics of bacteriophagesthat could pose a risk to human health and theenvironment. Several aspects that should be addressedwhen manipulating them in laboratories are discussed,with illustrations of relevant examples. Finally, basedon the risk assessment conclusion, biosafety recommendations(work practices, safety equipment, and wastemanagement) are proposed.</p

Le manuel de biosécurité. Canevas et guide de rédaction.2761

Ce document constitue un guide pratique pour les responsables de la biosécurité dans la rédaction du manuel de biosécurité. Il vise aussi à informer les autorités compétentes pour l&#039;autorisation et l&#039;inspection des laboratoires où sont manipulés des organismes génétiquement modifiés (OGM) ou pathogènes, sur le contenu du manuel de biosécurité. Le manuel de biosécurité s&#039;adresse au personnel scientifique (chercheurs, technologues de laboratoire) travaillant dans les laboratoires, il leur procure les informations, les lignes directrices et les procédures qui leur permettront de travailler de manière sûre en réduisant ou en éliminant les risques d&#039;exposition au danger biologique.</p

Biological risk assessment sheets : Practical examples of risk assessment and biosafety recommendations for the contained use of genetically modified (micro-)organisms

To ensure that all appropriate measures are taken to avoid adverse effects on human health and the environment that might arise from the contained use of genetically modified and/or pathogenic organisms, an risk assessment is carried out as regards to the biological risks. While classification list of pathogenic, non genetically modified, organisms provide a tool to assess the biological risks associated to the contained use of those organisms, the risk assessment of GMO appear to be more complex. This document provides a practical means for performing risk assessment of contained uses dealing with genetically modified organisms (GMOs), whether they are pathogenic or not. The first part offers a brief summary of the key issues of risk assessment of contained uses. The second part (annexes) consist of examples that have been elaborated to illustrate the risk assessment of contained uses dealing with GMOs.</p

Risk assessment of laboratories involving the manipulation of unconventional agents causing TSE

The present document aims at summarizing the biosafety recommendations and the containment level required for laboratories where animal and human tissues potentially contaminated by a TSE (Transmissible Spongiform Encephalitis) causing agent are manipulated. A particular attention will be paid to decontamination procedures, as the prion protein1 is remarkably resistant to conventional inactivation methods and may stay infectious for long periods of time. We will discuss large surface decontamination procedures of facilities handling TSE causing agents. This is of specific concern for laboratories that have been manipulating TSE causing agents, sometimes for years, but wish today dedicate the facilities to another activity