The temperate Salmonella enterica bacteriophage L is a close relative of the very well studied bacteriophage P22. In this study we show that the L procapsid assembly and DNA packaging genes, which encode terminase, portal, scaffold, and coat proteins, are extremely close relatives of the homologous P22 genes (96.3 to 99.1% identity in encoded amino acid sequence). However, we also identify an L gene, dec, which is not present in the P22 genome and which encodes a protein (Dec) that is present on the surface of L virions in about 150 to 180 molecules/virion. We also show that the Dec protein is a trimer in solution and that it binds to P22 virions in numbers similar to those for L virions. Its binding dramatically stabilizes P22 virions against disruption by a magnesium ion chelating agent. Dec protein binds to P22 coat protein shells that have expanded naturally in vivo or by sodium dodecyl sulfate treatment in vitro but does not bind to unexpanded procapsid shells. Finally, analysis of phage L restriction site locations and a number of patches of nucleotide sequence suggest that phages ST64T and L are extremely close relatives, perhaps the two closest relatives that have been independently isolated to date among the lambdoid phages.Bacteriophage L was reported to have been isolated after it was induced by UV irradiation from Salmonella enterica strain LT2 by Bezdek and Amati (2) in Czechoslovakia in 1967. Curiously, currently used LT2 cultures do not carry this prophage, although they do carry up to four other intact prophages (7, 40), so either extant LT2 strains have lost their L prophage or the strain used by those authors had inadvertently become lysogenized by phage L from the environment. Whatever its exact origin, L is a temperate, short-tailed doublestranded-DNA (dsDNA) bacteriophage that infects Salmonella enterica serovar Typhimurium (2). It is a rather close relative of the well-studied phage P22, with which it can form viable hybrids (2,3,23). It has immunity (C2 and Cro repressors), C1 activator, and replication protein specificities different from those of P22, but P22 regulatory genes c3, 23, and 24, virion assembly genes 1, 2, 3, 5, 8, 9, 10, 16, and 20, and lysis genes 13, 15, and 19 have been shown to be interchangeable between the two phages (2-4, 23, 31, 36, 51). Unlike P22, L does not have an immunity I region (27, 57) or a sieB function (58). DNA heteroduplex analysis (62) as well as physical map (27) and genetic map (56) construction showed that related genes are present in the same order on the P22 and L chromosomes. L virions are very similar in appearance to P22 virions when viewed in the electron microscope with negative staining (2), the two virions contain generally similar structural proteins (2, 27, 39), and their virion DNAs are indistinguishable in length (27). However, L virions have a significantly lighter equilibrium banding density in CsCl gradients than those of P22 (2), and phage L virions contain a major structural protein with a molecular mass of approximately 15 kDa tha...

A recent survey of insecticide resistance in two of the most problematic pests in UK glasshouses revealed some new developments. At least some individuals in all UK samples of Trialeurodes vaporariorum that were tested resisted the insect growth regulator (IGR) buprofezin. The most strongly resistant strains were unaffected by the field application rate of this compound, and even samples from populations that had never been exposed to buprofezin contained individuals that survived the highest concentration applied (10,000 mg litre-1). The field rate of buprofezin was shown to select for resistance through vapour action alone. The benzophenylurea teflubenzuron, an unrelated IGR, was cross-resisted by buprofezin-resistant individuals. There was no evidence of resistance to imidacloprid, but all T vaporariorum strains tested, regardless of origin, exhibited a high innate tolerance to nicotine, when compared with another whitefly species, Bemisia tabaci. Marked resistance to fenbutatin oxide and tebufenpyrad was found in single glasshouse populations of Tetranychus urticae, but these compounds and abamectin appeared to remain highly effective against all other strains collected.

Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.

BackgroundWhile the molecular mechanisms of DNA-protein specificity at the origin of replication have been determined in many model organisms, these interactions remain unknown in the majority of higher eukaryotes and numerous vertebrate viruses. Similar to many viral origins of replication, adeno-associated virus (AAV) utilizes a cis-acting origin of replication and a virus specific Replication protein (Rep) to faithfully carry out self-priming replication. The mechanisms of AAV DNA replication are generally well understood. However, the molecular basis of specificity between the Rep protein and the viral origin of replication between different AAV serotypes remains uncharacterized.Methodology/Principal FindingsBy generating a panel of chimeric and mutant origins between two AAV serotypes, we have mapped two independent DNA-Protein interfaces involved in replicative specificity. In vivo replication assays and structural modeling demonstrated that three residues in the AAV2 Rep active site are necessary to cleave its cognate origin. An analogous origin (AAV5) possesses a unique interaction between an extended Rep binding element and a 49 aa region of Rep containing two DNA binding interfaces.Conclusions/SignificanceThe elucidation of these structure-function relationships at the AAV origin led to the creation of a unique recombinant origin and compatible Rep protein with properties independent of either parent serotype. This novel origin may impact the safety and efficacy of AAV as a gene delivery tool. This work may also explain the unique ability of certain AAV serotypes to achieve site-directed integration into the human chromosome. Finally, this result impacts the study of conserved DNA viruses which employ rolling circle mechanisms of replication.

Quantitative genomic and proteomic evaluation of human latent fingerprint depositions represents a challenge within the forensic field, due to the high variability in the amount of DNA and protein initially deposited. To better assess recovery techniques for touch depositions, we present a method to produce simple and customizable artificial fingerprints. These artificial fingerprint samples include the primary components of a typical latent fingerprint, specifically sebaceous fluid, eccrine perspiration, extracellular DNA, and proteinaceous epidermal skin material (i.e., shed skin cells). A commercially available emulsion of sebaceous and eccrine perspiration material provides a chemically-relevant suspension solution for fingerprint deposition, simplifying artificial fingerprint production. Extracted human genomic DNA is added to accurately mimic the extracellular DNA content of a typical latent print and comparable DNA yields are recovered from the artificial prints relative to human prints across surface types. Capitalizing on recent advancements in the use of protein sequence identification for human forensic analysis, these samples also contain a representative quantity of protein, originating from epidermal skin cells collected from the fingers and palms of volunteers. Proteomic sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicates a high level of protein overlap between artificial and latent prints. Data are available via ProteomeXchange with identifier PXD015445. By including known quantities of DNA and protein into each artificial print, this method enables total DNA and protein recovery to be quantitatively assessed across different sample collection and extraction methods to better evaluate extraction efficiency. Collectively, these artificial fingerprint samples are simple to make, highly versatile and customizable, and accurately represent the biochemical composition and biological signatures of human fingerprints.