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  • 1
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    Screening of KHP30-like prophages among Japanese Helicobacter pylori strains, and genetic analysis of a defective KHP30-like prophage sequence integrated in the genome of the H. pylori strain NY40 (2016)
    Oxford University Press
    Details
    Publication Date: 2016-07-24
    Description: We have recently reported the active Helicobacter pylori bacteriophages (phages), KHP30 and KHP40, the genomic DNAs of which exist as episomes in host bacterial strains isolated in Japan (i.e. pseudolysogeny). In this study, we examined the possibility of the lysogeny of active KHP30-like phages in Japanese H. pylori strains, because their genomes contain a putative integrase gene. Only the NY40 strain yielded partial detection of a KHP30-like prophage sequence in PCR among 174 Japanese H. pylori isolates, except for strains producing the above active phages. Next, according to the genomic analysis of the NY40 strain, the KHP30-like prophage sequence was found to be located from ca. 524 to 549 kb in the host chromosome. The attachment sites, attL and attR , in the NY40 genome showed almost the same genomic location and sequence as those detected in a French isolate B38, suggesting that an active parental KHP30-like phage had integrated into the ancestral NY40 genome in a site-specific manner. The prophage found in the NY40 genome was assumed to have been genetically modified, after site-specific integration. These, together with the data in the KHP30-like prophages of other H. pylori genomes, suggest that the lysogenic state of the KHP30-like phages is generally unstable.
    Keywords: Virology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Vitamin C induced DevR-dependent synchronization of Mycobacterium smegmatis growth and its effect on the proliferation of mycobacteriophage D29 (2016)
    Oxford University Press
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    Publication Date: 2016-05-12
    Description: Vitamin C is known to inhibit mycobacterial growth by acting as a hypoxia inducing agent. While investigating how mycobacteriophage growth is influenced by hypoxic conditions induced by vitamin C, using Mycobacterium smegmatis - mycobacteriophage D29 as a model system, it was observed that prior exposure of the host to such conditions resulted in increased burst size of the phage. Vitamin C pre-exposure was also found to induce synchronous growth of the host. A mutant defective in DevR, the response regulator that controls hypoxic responses in mycobacteria, neither supported higher phage bursts nor was it able to undergo synchronized growth following vitamin C pre-exposure, indicating thereby that the two phenomena are interrelated. Further evidence supporting such an interrelationship was obtained from the observation that phage burst sizes varied depending on the stage of synchronous growth that the host cells were in, at the time of infection—higher bursts were observed in the resting/synthetic phases and lower in the dividing ones. The effects were specific in nature as synchronization by an unrelated method, known as ‘crowding’, did not lead to the same consequence. The results indicate that growth synchronization induced by vitamin C treatment is a DevR-dependent phenomenon which is exploited by mycobacteriophage D29 to grow in larger numbers.
    Keywords: Virology
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    Topics: Biology
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  • 3
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    Single-stranded DNA phages: from early molecular biology tools to recent revolutions in environmental microbiology (2016)
    Oxford University Press
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    Publication Date: 2016-03-05
    Description: Single-stranded DNA (ssDNA) phages are profoundly different from tailed phages in many aspects including the nature and size of their genome, virion size and morphology, mutation rate, involvement in horizontal gene transfer, infection dynamics and cell lysis mechanisms. Despite the importance of ssDNA phages as molecular biology tools and model systems, the environmental distribution and ecological roles of these phages have been largely unexplored. Viral metagenomics and other culture-independent viral diversity studies have recently challenged the perspective of tailed, double-stranded DNA (dsDNA) phages, dominance by demonstrating the prevalence of ssDNA phages in diverse habitats. However, the differences between ssDNA and dsDNA phages also substantially limit the efficacy of simultaneously assessing the abundance and diversity of these two phage groups. Here we provide an overview of the major differences between ssDNA and tailed dsDNA phages that may influence their effects on bacterial communities. Furthermore, through the analysis of 181 published metaviromes we demonstrate the environmental distribution of ssDNA phages and present an analysis of the methodological biases that distort their study through metagenomics.
    Keywords: Virology
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  • 4
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    Experimental evidence for proteins constituting virion components and particle morphogenesis of bacteriophage ZF40 (2016)
    Oxford University Press
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    Publication Date: 2016-03-09
    Description: Bacteriophage ZF40 is the only currently available, temperate Myoviridae phage infecting the potato pathogen Pectobacterium carotovorum subsp . carotovorum . Despite its unusual tail morphology, its major tail sheath and tube proteins remained uncharacterized after the initial genome annotation. Using ESI tandem mass-spectrometry, 24 structural proteins of the ZF40 virion were identified, with a sequence coverage ranging between 15.8% and 87.8%. The putative function of 16 proteins could be elucidated based on secondary structure analysis and conservative domain searches. The experimental annotation of 35% of the encoded gene products within the structural region of the genome represents a complete view of the virion structure, which can serve as the basis for future structural analysis as a model phage.
    Keywords: Virology
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    Topics: Biology
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  • 5
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    Host receptors for bacteriophage adsorption (2016)
    Oxford University Press
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    Publication Date: 2016-02-07
    Description: The adsorption of bacteriophages (phages) onto host cells is, in all but a few rare cases, a sine qua non condition for the onset of the infection process. Understanding the mechanisms involved and the factors affecting it is, thus, crucial for the investigation of host–phage interactions. This review provides a survey of the phage host receptors involved in recognition and adsorption and their interactions during attachment. Comprehension of the whole infection process, starting with the adsorption step, can enable and accelerate our understanding of phage ecology and the development of phage-based technologies. To assist in this effort, we have established an open-access resource—the Phage Receptor Database (PhReD)—to serve as a repository for information on known and newly identified phage receptors.
    Keywords: Virology
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  • 6
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    Production and characterization of a Brazilian candidate antigen for Hepatitis E Virus genotype 3 diagnosis (2016)
    Oxford University Press
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    Publication Date: 2016-02-25
    Description: Hepatitis E, caused by hepatitis E virus (HEV), is a viral infectious pathology of great importance in the public health. Hepatitis E outbreaks were registered in developing countries with poor or no sanitation, where drinking water was contaminated with fecal material, but also in many industrialized countries probably due to consumption of HEV-positive swine meat. In this study, we present the development and characterization of a recombinant antigen from ORF2 HEV genotype 3. Viral RNA was extracted from swine feces infected with the native virus. A total of 267 residues from the C-terminal ORF2 (394–661) coding sequence were cloned into the pET20a vector and expressed in Escherichia coli ER2566. Recombinant protein was purified by liquid chromatography and the fragment obtained a 98% homology against other human or swine HEV genotype 3 ORF2 sequences. Wistar rats were inoculated with ORF2p, developing antibodies able to recognize both the homologous antigen and the native HEV genotype 3 ORF2 present in infected stool. In parallel, HEV-negative swine were experimentally challenged with HEV genotype 3. ORF2 was detected by PCR 14 days post-inoculation in three-fourth piglets’ feces and one week later by dot blot. In conclusion, this study proved the immunogenic and antigenic properties of the recombinant protein ORF2p.
    Keywords: Virology
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  • 7
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    Diversity of phage infection types and associated terminology: the problem with 'Lytic or lysogenic (2016)
    Oxford University Press
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    Publication Date: 2016-03-24
    Description: Bacteriophages, or phages, are viruses of members of domain Bacteria. These viruses play numerous roles in shaping the diversity of microbial communities, with impact differing depending on what infection strategies specific phages employ. From an applied perspective, these especially are communities containing undesired or pathogenic bacteria that can be modified through phage-mediated bacterial biocontrol, that is, through phage therapy. Here we seek to categorize phages in terms of their infection strategies as well as review or suggest more descriptive, accurate or distinguishing terminology. Categories can be differentiated in terms of (1) whether or not virion release occurs (productive infections versus lysogeny, pseudolysogeny and/or the phage carrier state), (2) the means of virion release (lytic versus chronic release) and (3) the degree to which phages are genetically equipped to display lysogenic cycles (temperate versus non-temperate phages). We address in particular the use or overuse of what can be a somewhat equivocal phrase, ‘Lytic or lysogenic’, especially when employed as a means of distinguishing among phages types. We suggest that the implied dichotomy is inconsistent with both modern as well as historical understanding of phage biology. We consider, therefore, less ambiguous terminology for distinguishing between ‘Lytic’ versus ‘Lysogenic’ phage types.
    Keywords: Virology
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  • 8
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    Computational prospecting the great viral unknown (2016)
    Oxford University Press
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    Publication Date: 2016-04-24
    Description: Bacteriophages play an important role in host-driven biological processes by controlling bacterial population size, horizontally transferring genes between hosts and expressing host-derived genes to alter host metabolism. Metagenomics provides the genetic basis for understanding the interplay between uncultured bacteria, their phage and the environment. In particular, viral metagenomes (viromes) are providing new insight into phage-encoded host genes (i.e. auxiliary metabolic genes; AMGs) that reprogram host metabolism during infection. Yet, despite deep sequencing efforts of viral communities, the majority of sequences have no match to known proteins. Reference-independent computational techniques, such as protein clustering, contig spectra and ecological profiling are overcoming these barriers to examine both the known and unknown components of viromes. As the field of viral metagenomics progresses, a critical assessment of tools is required as the majority of algorithms have been developed for analyzing bacteria. The aim of this paper is to offer an overview of current computational methodologies for virome analysis and to provide an example of reference-independent approaches using human skin viromes. Additionally, we present methods to carefully validate AMGs from host contamination. Despite computational challenges, these new methods offer novel insights into the diversity and functional roles of phages in diverse environments.
    Keywords: Virology
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  • 9
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    Intrinsic characteristics of Min proteins on the cell division of Helicobacter pylori (2016)
    Oxford University Press
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    Publication Date: 2016-03-04
    Description: Helicobacter pylori divides in the human stomach resulting in persistent infections and causing various disorders. Bacterial cell division is precisely coordinated by many molecules, including FtsZ and Min proteins. However, the role of Min proteins in H. pylori division is poorly understood. We investigated the functional characteristics of Min proteins in wild-type HPK5 and five HPK5-derivative mutants using morphological and genetic approaches. All mutants showed a filamentous shape. However, the bacterial cell growth and viability of three single-gene mutants ( minC , minD , minE ) were similar to that of the wild-type. The coccoid form number was lowest in the minE -disruptant, indicating that MinE contributes to the coccoid form conversion during the stationary phase. Immunofluorescence microscopic observations showed that FtsZ was dispersedly distributed throughout the bacterial cell irrespective of nucleoid position in only minD -disruptants, indicating that MinD is involved in the nucleoid occlusion system. A chase assay demonstrated that MinC loss suppressed FtsZ-degradation, indicating that FtsZ degrades in a MinC-dependent manner. Molecular interactions between FtsZ and Min proteins were confirmed by immunoprecipitation (IP)-western blotting (WB), suggesting the functional cooperation of these molecules during bacterial cell division. This study describes the intrinsic characteristics of Min proteins and provides new insights into H. pylori cell division.
    Keywords: Virology
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  • 10
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    Biogeography of bacteriophages at four hydrothermal vent sites in the Antarctic based on g23 sequence diversity (2016)
    Oxford University Press
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    Publication Date: 2016-03-13
    Description: In this study, which was carried out within the ChEsSO consortium project (Chemosynthetically driven ecosystems south of the Polar Front), we sampled two hydrothermal vent sites on the East Scotia Ridge, Scotia Sea, one in the Kemp Caldera, South Sandwich Arc and one in the Bransfield Strait, north-west of the Antarctic Peninsula, which exhibit strong differences in their chemical characteristics. We compared a subset of their bacteriophage population by Sanger- and 454-sequencing of g23 , which codes for the major capsid protein of T4likeviruses. We found that the sites differ vastly in their bacteriophage diversity, which reflects the differences in the chemical conditions and therefore putatively the differences in microbial hosts living at these sites. Comparing phage diversity in the vent samples to other aquatic samples, the vent samples formed a distinct separate cluster, which also included the non-vent control samples that were taken several hundred meters above the vent chimneys. This indicates that the influence of the vents on the microbial population and therefore also the bacteriophage population extends much further than anticipated.
    Keywords: Virology
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  • 11
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    Protective role of host aquaporin 6 against Hazara virus, a model for Crimean-Congo hemorrhagic fever virus infection (2016)
    Oxford University Press
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    Publication Date: 2016-04-08
    Description: Crimean–Congo hemorrhagic fever virus (CCHFV) is an arthropod-borne pathogen that causes infectious disease with severe hemorrhagic manifestations in vascular system in humans. The proper function of the cells in the vascular system is critically regulated by aquaporins (AQP), water channels that facilitate fluxes of water and small solutes across membranes. With Hazara virus as a model for CCHFV, we investigated the effects of viruses on AQP6 and the impact of AQP6 on virus infectivity in host cells, using transiently expressed GFP-AQP6 cells, immunofluorescent assay for virus detection, epifluorescent imaging of living cells and confocal microscopy. In GFP-AQP6 expressing cells, Hazara virus reduced both the cellular and perinuclear AQP6 distribution and changed the cell area. Infection of human cell with CCHFV strain IbAR 10200 downregulated AQP6 expression at mRNA level. Interestingly, the overexpression of AQP6 in host cells decreased the infectivity of Hazara virus, speaking for a protective role of AQP6. We suggest the possibility for AQP6 being a novel player in the virus–host interactions, which may lead to less severe outcomes of an infection.
    Keywords: Virology
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  • 12
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    A novel prophage lysin Ply5218 with extended lytic activity and stability against Streptococcus suis infection (2016)
    Oxford University Press
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    Publication Date: 2016-09-14
    Description: Streptococcus suis ( S. suis ) is an emerging zoonotic agent that exhibits high level resistance to classic antibiotics and a heavy burden in the swine industry. Therefore alternative antibacterial agents need to be developed. A novel endolysin derived from the S. suis temperate phage phi5218, termed Ply5218, was identified. The minimum inhibitory concentration (MIC) of Ply5218 was 2.5 μg ml –1 against S. suis strain HA9801, an activity many times greater than the lysins reported previously (MIC of LY7917 and Ply30 against HA9801 were 80 and 64 μg ml –1 , respectively). Ply5218 at 10 μg ml –1 in vitro exerted broad antibacterial activities against S. suis strains with OD 600 ratios decreased from 1 to 〈0.2 within 1 h. Moreover, Ply5218 showed favorable thermal stability. It was stable at 50°C 〉30 min, 4°C 〉30 days, –80°C 〉7 months, and 〉60% of the enzyme activity remained after 5 min pre-incubation at 70°C. In vivo , a 0.2 mg dose of Ply5218 protected 90% (9/10) of mice after infection with S. suis HA9801. Finally, Ply5218 maintained high antibacterial activity in some bio-matrices, such as culture media and milk. The data indicate that Ply5218 has all the characteristics to be an effective therapeutic agent against multiple S. suis infections .
    Keywords: Virology
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  • 13
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    Incidence of diverse dsRNA mycoviruses in Trichoderma spp. causing green mold disease of shiitake Lentinula edodes (2016)
    Oxford University Press
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    Publication Date: 2016-10-12
    Description: A total of 315 fungal isolates causing green mold disease were collected from contaminated artificial logs and sawdust bags used for cultivating shiitake Lentinula edodes in Korea and were analyzed for the presence of double-stranded RNA (dsRNA). dsRNA, which was purified using dsRNA-specific chromatography and verified by dsRNA-specific RNaseIII digestion, was detected in 32 isolates. The molecular taxonomy of dsRNA-infected isolates indicated that all isolates belonged to the Trichoderma spp.. The number and size of dsRNAs varied among isolates and the band patterns could be categorized into 15 groups. Although there were seven dsRNA groups observed in multiple isolates, eight groups were found to occur in single isolates. The most common dsRNA group, group VI, which contained a band of 10 kb, occurred in 10 isolates encompassing three species of Trichoderma . Partial sequence analysis of two selected dsRNA groups revealed a high degree of similarity to sequences of a RNA-dependent RNA polymerase, hypothetical protein and polyprotein genes of other hypoviruses such as Macrophomina phaseolina hypovirus 1, Trichoderma hypovirus, and Fusarium graminearum hypovirus 2, respectively, indicating the occurrence of mycoviruses in Trichoderma spp.. Northern blot analysis suggested that many different mycoviruses, which have not been identified yet, exist in Trichoderma .
    Keywords: Virology
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  • 14
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    Subinhibitory concentrations of punicalagin reduces expression of virulence-related exoproteins by Staphylococcus aureus (2016)
    Oxford University Press
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    Publication Date: 2016-12-16
    Description: Staphylococcus aureus produces a number of virulence factors. The major virulence factors exhibited by S . aureus include various antigens, enzymes, cytotoxins and exotoxins (e.g. hemolysins, enterotoxins and toxic shock syndrome toxin). In this report, we show the influence of punicalagin on the secretion of exoprotein from S . aureus by western blotting, tumor necrosis factor (TNF) release assay and quantitative RT-PCR. When added to S . aureus cultures at an OD600 of 0.9, graded subinhibitory concentrations of punicalagin reduced the production of α -toxin, SEA and SEB in methicillin-resistant Staphylococcus aureus in a dose-dependent manner. Consistently, punicalagin reduced TNF-inducing activity by S . aureus culture supernatants. Here, the transcriptional level of agr (accessory gene regulator) in S . aureus was inhibited by punicalagin, suggesting that the reduced transcription may affect the secretion of exotoxins. These findings suggest that the expression of α -toxin and enterotoxins in S . aureus is sensitive to the action of punicalagin, which may be an advantageous candidate in the treatment of toxigenic staphylococcal disease.
    Keywords: Virology
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  • 15
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    Genomic diversity of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum (2016)
    Oxford University Press
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    Publication Date: 2016-12-29
    Description: Bacteriophages infecting the fish pathogen Flavobacterium psychrophilum can potentially be used to prevent and control outbreaks of this bacterium in salmonid aquaculture. However, the application of bacteriophages in disease control requires detailed knowledge on their genetic composition. To explore the diversity of F. pyschrophilum bacteriophages, we have analyzed the complete genome sequences of 17 phages isolated from two distant geographic areas (Denmark and Chile), including the previously characterized temperate bacteriophage 6H. Phage genome size ranged from 39 302 to 89 010 bp with a G+C content of 27%–32%. None of the bacteriophages isolated in Denmark contained genes associated with lysogeny, whereas the Chilean isolates were all putative temperate phages and similar to bacteriophage 6H. Comparative genome analysis showed that phages grouped in three different genetic clusters based on genetic composition and gene content, indicating a limited genetic diversity of F. psychrophilum -specific bacteriophages. However, amino acid sequence dissimilarity (25%) was found in putative structural proteins, which could be related to the host specificity determinants. This study represents the first analysis of genomic diversity and composition among bacteriophages infecting the fish pathogen F. psychrophilum and discusses the implications for the application of phages in disease control.
    Keywords: Virology
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  • 16
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    Phage vB_BsuP-Goe1: the smallest identified lytic phage of Bacillus subtilis (2016)
    Oxford University Press
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    Publication Date: 2016-10-08
    Description: Currently, interest in phage science is on the rise again as this subject remains largely unexplored, and its potential diversity mainly untapped. Here, we present phage vB_BsuP-Goe1, a new isolate that infects Bacillus subtilis . The phage forms round plaques with a matt outline on agar plates containing a B. subtilis 6 lawn. Transmission electron microscopy and genomic analyses revealed that phage vB_BsuP-Goe1 belongs to the Podoviridae family as a new member of the Phi29likevirus genus. The phage genome comprises 18 379 bp that encode for one RNA gene and for 24 open reading frames. Phage vB_BsuP-Goe1 clusters with phage B103 and Nf within the Phi29likevirus genus and is currently the smallest member of this genus. Genome comparison with members of the Phi29likevirus genus reveals a conserved gene set coding for essential replication and morphogenesis functions while the genome extremities are more prone to gene and genome organisation variability.
    Keywords: Virology
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