Bacteriophage Φ29 Research Articles

Selection of surrogate viruses for process control in detection of SARS-CoV-2 in wastewater

Since SARS-CoV-2 RNA in wastewater is often present at low concentration or under detection limit, ensuring the reliability of detection processes using appropriate process controls is essential. The objective of this study was to evaluate applicability and limitations of candidate surrogate viruses as process controls under combinations of different virus concentration and RNA extraction methods. Detection efficiency of SARS-CoV-2 spiked in wastewater was compared with those of candidate surrogate viruses of bacteriophage ϕ6, pepper mild mottle virus (PMMoV), F-specific coliphage (F-phage), and murine norovirus (MNV). After inactivated SARS-CoV-2 and ϕ6 were spiked in two different wastewaters, the viruses in solid and liquid fractions of wastewater were concentrated by centrifuge and polyethylene glycol (PEG) precipitation, respectively. Viral RNA was extracted by using QIAamp Viral RNA Mini Kit and 3 other commercially available extraction kits, then quantified by reverse transcription-quantitative PCR using CDCN1 assay. Regardless of extraction kits, SARS-CoV-2 was consistently detected with good efficiency from both liquid (11–200%) and solid fractions (7.1–93%). Among the candidate process controls, PMMoV was widely detected at good efficiencies from both liquid and solid fractions regardless of selection of RNA extraction kits. F-phage and MNV also showed good detection efficiencies in most combinations of wastewater fractions and RNA extraction kits. An enveloped virus ɸ6 was found often undetected or to have very low detection efficiency (0.1–4.2%) even when SARS-CoV-2 spiked in wastewater was detected with good efficiency. Consequently, PMMoV is widely applicable as process control for detection of SARS-CoV-2 either in liquid fractions concentrated by PEG precipitation, or in solid fractions concentrated by centrifuge.

Isolation of the Novel Phage PHB09 and Its Potential Use against the Plant Pathogen Pseudomonas syringae pv. actinidiae.

Bacteriophages are viruses that specifically infect target bacteria. Recently, bacteriophages have been considered potential biological control agents for bacterial pathogens due to their host specificity. Pseudomonas syringae pv. actinidiae (Psa) is a reemerging pathogen that causes bacterial canker of kiwifruit (Actinidia sp.). The economic impact of this pest and the development of resistance to antibiotics and copper sprays in Psa and other pathovars have led to investigation of alternative management strategies. Phage therapy may be a useful alternative to conventional treatments for controlling Psa infections. Although the efficacy of bacteriophage φ6 was evaluated for the control of Psa, the characteristics of other DNA bacteriophages infecting Psa remain unclear. In this study, the PHB09 lytic bacteriophage specific to Psa was isolated from kiwifruit orchard soil. Extensive host range testing using Psa isolated from kiwifruit orchards and other Pseudomonas strains showed PHB09 has a narrow host range. It remained stable over a wide range of temperatures (4–50 °C) and pH values (pH 3–11) and maintained stability for 50 min under ultraviolet irradiation. Complete genome sequence analysis indicated PHB09 might belong to a new myovirus genus in Caudoviricetes. Its genome contains a total of 94,844 bp and 186 predicted genes associated with phage structure, packaging, host lysis, DNA manipulation, transcription, and additional functions. The isolation and identification of PHB09 enrich the research on Pseudomonas phages and provide a promising biocontrol agent against kiwifruit bacterial canker.

Comparison of virus concentration methods for the reverse-transcriptase quantitative PCR (RT-qPCR)-based detection of bacteriophage Φ6, an enveloped virus surrogate for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, from untreated wastewater at St. George’s University (SGU), Grenada

Introduction/ObjectiveWith the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the consequent opening of borders, it is of paramount importance to monitor communities for the presence of potential COVID-19 clusters. Wastewater-based epidemiology (WBE) is an effective way to monitor community spread of pathogens and has been implemented in many countries to identify potential outbreaks. Most standardized methodologies used in WBE for the detection of viruses have been validated for non-enveloped viruses; therefore, there is a need for the creation of a standardized method for the detection of enveloped viruses, such as SARS-CoV-2.Methods/Case ReportWastewater seeded with a Pseudomonas syringae bacteriophage (Φ6) was used to test the efficiency of five wastewater virus concentration methods. Two methods were based on AlCl3 flocculation with different pH adjustments. The other methods utilized membrane filtration followed by beef elution: one added a pretreatment with HCl; another added a pretreatment with MgCl2; and finally, an untreated control. A known amount of Bacteriophage Φ6 was used for all methods, and detection was quantified by reverse-transcription quantitative polymerase chain reaction.Results (if a Case Study enter NA)The Cq values for each method, ranging from 24.38–38.6, were compared. The highest recovery of Φ6 was obtained with the AlCl3 flocculation method with an adjusted pH of 6, followed in descending order by MgCl2-pretreated sample with beef elution; HCl-pretreated sample with beef elution, the untreated sample, and AlCl3 flocculation method with an adjusted pH of 3.5.ConclusionThe results suggest that AlCl3 flocculation with an adjusted pH of 6 may be a quick and cost-effective methodology for the detection of enveloped viruses in WBE. This can facilitate the implementation of WBE at SGU, Grenada, as a useful tool to conduct active surveillance and track outbreaks of enveloped viral diseases, such as SARS-CoV-2.

Formation and Repair of an Interstrand DNA Cross-Link Arising from a Common Endogenous Lesion.

Interstrand DNA cross-links (ICLs) are cytotoxic because they block the strand separation required for read-out and replication of the genetic information in duplex DNA. The unavoidable formation of ICLs in cellular DNA may contribute to aging, neurodegeneration, and cancer. Here, we describe the formation and properties of a structurally complex ICL derived from an apurinic/apyrimidinic (AP) site, which is one of the most common endogenous lesions in cellular DNA. The results characterize a cross-link arising from aza-Michael addition of the N2-amino group of a guanine residue to the electrophilic sugar remnant generated by spermine-mediated strand cleavage at an AP site in duplex DNA. An α,β-unsaturated iminium ion is the critical intermediate involved in ICL formation. Studies employing the bacteriophage φ29 polymerase provided evidence that this ICL can block critical DNA transactions that require strand separation. The results of biochemical studies suggest that this complex strand break/ICL might be repaired by a simple mechanism in which the 3'-exonuclease action of the enzyme apurinic/apyrimidinic endonuclease (APE1) unhooks the cross-link to initiate repair via the single-strand break repair pathway.

Antiviral Face Mask Functionalized with Solidified Hand Soap: Low-Cost Infection Prevention Clothing against Enveloped Viruses Such as SARS-CoV-2.

Infection prevention clothing is becoming an essential protective tool in the current pandemic, especially because now we know that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can easily infect humans in poorly ventilated indoor spaces. However, commercial infection prevention clothing is made of fabrics that are not capable of inactivating the virus. Therefore, viral infections of symptomatic and asymptomatic individuals wearing protective clothing such as masks can occur through aerosol transmission or by contact with the contaminated surfaces of the masks, which are suspected as an increasing source of highly infectious biological waste. Herein, we report an easy fabrication method of a novel antiviral non-woven fabric containing polymer filaments that were coated with solidified hand soap. This extra protective fabric is capable of inactivating enveloped viruses such as SARS-CoV-2 and phage Φ6 within 1 min of contact. In this study, this antiviral fabric was used to fabricate an antiviral face mask and did not show any cytotoxic effect in human keratinocyte HaCaT cells. Furthermore, this antiviral non-woven fabric could be used for the fabrication of other infection prevention clothing such as caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons, and shoe covers. Therefore, this low-cost technology could provide a wide range of infection-protective tools to combat COVID-19 and future pandemics in developed and underdeveloped countries.

Detection of SARS-CoV-2 in wastewater is influenced by sampling time, concentration method, and target analyzed.

The detection of SARS-CoV in wastewater has been proposed as a tool for monitoring COVID-19 at the community level. Although many reports have been published about detecting viral RNA in wastewater and its presence has been linked to infected people, appropriate analytical methodologies to use this approach have not yet been established. In this study, we compared ultrafiltration, polyethylene glycol precipitation, flocculation using AlCl3, and flocculation with skim milk for the recovery of SARS-CoV-2, using RNA from patients with positive diagnoses for COVID-19 and Pseudomonas phage φ6 as the control. We also evaluated the primers for detecting the E, RdRp, and N genes of the virus, as well as different storage times. Differences in the recovery efficiencies were evident with the different concentration methods, the best being ultrafiltration and precipitation with aluminum, which had recovery rates of 42.0% and 30.0%, respectively, when virus was present at high levels. Significant differences were found between the recoveries using wastewater and deionized water and between different storage times, with better recoveries for 6 and 12 h samplings. The E gene was the only one detected in all the samples analyzed. The results show that although this approach can provide important data for studying the pandemic, clear protocols are necessary for investigations to be comparable.

A DNA packaging motor inchworms along one strand allowing it to adapt to alternative double-helical structures

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport ordered, helical substrates, such as the bacteriophage ϕ29 dsDNA packaging motor. This pentameric motor alternates between an ATP loading dwell and a hydrolysis burst wherein it packages one turn of DNA in four steps. When challenged with DNA-RNA hybrids and dsRNA, the motor matches its burst to the shorter helical pitches, keeping three power strokes invariant while shortening the fourth. Intermittently, the motor loses grip on the RNA-containing substrates, indicating that it makes optimal load-bearing contacts with dsDNA. To rationalize these observations, we propose a helical inchworm translocation mechanism in which, during each cycle, the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

A Simple Electrostatic Precipitator for Trapping Virus Particles Spread via Droplet Transmission.

The purpose of this study was to develop a simple electrostatic apparatus to precipitate virus particles spread via droplet transmission, which is especially significant in the context of the recent coronavirus disease 2019 (COVID-19) pandemic. The bacteriophage φ6 of Pseudomonas syringae was used as a model of the COVID-19 virus because of its similar structure and safety in experiments. The apparatus consisted of a spiked, perforated stainless plate (S-PSP) linked to a direct-current voltage generator to supply negative charge to the spike tips and a vessel with water (G-water) linked to a ground line. The S-PSP and G-water surface were paralleled at a definite interval. Negative charge supplied to the spike tips positively polarised the G-water by electrostatic induction to form an electric field between them in which ionic wind and negative ions were generated. Bacteriophage-containing water was atomised with a nebuliser and introduced into the electric field. The mist particles were ionised by the negative ions and attracted to the opposite pole (G-water). This apparatus demonstrated a prominent ability to capture phage-containing mist particles of the same sizes as respiratory droplets and aerosols regardless of the phage concentration of the mist particles. The trapped phages were successfully sterilised using ozone bubbling. Thus, the present study provides an effective system for eliminating droplet transmission of viral pathogens from public spaces.

An automated room disinfection system using ozone is highly active against surrogates for SARS-CoV-2

BackgroundThe presence of coronaviruses on surfaces in the patient environment is a potential source of indirect transmission. Manual cleaning and disinfection measures do not always achieve sufficient removal of surface contamination. This increases the importance of automated solutions in the context of final disinfection of rooms in the hospital setting. Ozone is a highly effective disinfectant which, combined with high humidity, is an effective agent against respiratory viruses. Current devices allow continuous nebulization for high room humidity as well as ozone production without any consumables.AimIn the following study, the effectiveness of a fully automatic room decontamination system based on ozone was tested against bacteriophage Φ6 (phi 6) and bovine coronavirus L9, as surrogate viruses for the pandemic coronavirus SARS-CoV-2.MethodsFor this purpose, various surfaces (ceramic tile, stainless steel surface and furniture board) were soiled with the surrogate viruses and placed at two different levels in a gas-tight test room. After using the automatic decontamination device according to the manufacturer's instructions, the surrogate viruses were recovered from the surfaces and examined by quantitative cultures. Then, reduction factors were calculated.FindingsThe ozone-based room decontamination device achieved virucidal efficacy (reduction factor >4 log10) against both surrogate organisms regardless of the different surfaces and positions confirming a high activity under the used conditions.ConclusionOzone is highly active against SARS-CoV-2 surrogate organisms. Further investigations are necessary for a safe application and efficacy in practice as well as integration into routine processes.

Structural Insights into gp16 ATPase in the Bacteriophage ϕ29 DNA Packaging Motor.

Biological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. Gene product 16 (gp16) in bacteriophage ϕ29 is among the most powerful biomotors known, which adopts a multisubunit ring-shaped structure and hydrolyzes ATP to package double-stranded DNA (dsDNA) into a preformed procapsid. Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment and molecular dynamics simulations revealed an essential binding surface of gp16-CTD for prohead RNA, a unique component of the motor complex. Furthermore, our simulations highlighted a dynamic interplay between the N-terminal domain and the CTD of gp16, which may play a role in driving movement of DNA into the procapsid. Lastly, we assembled an atomic structural model of the complete ϕ29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage ϕ29 and suggested how the individual domains of gp16 work together to power such translocation.

Preparation of cerium molybdates and their antiviral activity against bacteriophage Φ6 and SARS-CoV-2

Two cerium molybdates (Ce2Mo3O12 and γ-Ce2Mo3O13) were prepared using either polymerizable complex method or hydrothermal process. The obtained powders were almost single-phase with different cerium valence. Both samples were found to have antiviral activity against bacteriophage Φ6. Especially, γ-Ce2Mo3O13 exhibited high antiviral activity against both bacteriophage Φ6 and SARS-CoV-2 coronavirus, which causes COVID-19. A synergetic effect of Ce and molybdate ion was inferred along with the specific surface area as key factors for antiviral activity.

Isolation and Characterization of Bacillus cereus Phage vB_BceP-DLc1 Reveals the Largest Member of the Φ29-Like Phages.

Bacillus phage φ29 and its relatives have been considered as one of the most important model organisms for DNA replication, transcription, morphogenesis, DNA packaging studies, and nanotechnology applications. Here, we isolated and characterized a new member of the φ29-like phage, named Bacillus cereus phage vB_BceP-DLc1. This phage, with a unique inserted gene cluster, has the largest genome among known φ29-like phages. DLc1 can use the surface carbohydrate structures of the host cell as receptors and only infects the most related B. cereus strains, showing high host-specificity. The adsorption rate constant and life cycle of DLc1 under experimental conditions were also determined. Not only stable under temperatures below 55 °C and pH range from 5 to 11, the new phage also showed tolerance to high concentrations of NaCl, 75% ethanol, chloroform, and mechanical vortex, which is preferable for practical use in the food and pharmaceutical industries.

Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater

The primary concentration and molecular process are critical to implement wastewater-based epidemiology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the previously developed methods were optimized for nonenveloped viruses. Few studies evaluated if the methods are applicable to the efficient recovery of enveloped viruses from various types of raw sewage. This study aims (1) to compare the whole process recovery of Pseudomonas phage φ6, a surrogate for enveloped viruses, among combinations of primary concentration [ultrafiltration (UF), electronegative membrane vortex (EMV), and polyethylene glycol precipitation (PEG)] and RNA extraction methods (spin column-based method using QIAamp Viral RNA Mini Kit and acid guanidinium thiocyanate–phenol–chloroform extraction using TRIzol reagent) for three types of raw sewage and (2) to test the applicability of the method providing the highest φ6 recovery to the detection of SARS-CoV-2 RNA. Among the tested combinations, PEG+TRIzol provided the highest φ6 recovery ratio of 29.8% to 49.8% (geometric mean). UF + QIAamp Viral RNA Mini Kit provided the second highest φ6 recovery of 6.4% to 35.8%. The comparable φ6 recovery was observed for UF + TRIzol (13.8–30.0%). PEG + QIAamp Viral RNA Mini Kit provided only 1.4% to 3.0% of φ6 recovery, while coliphage MS2, a surrogate for nonenveloped viruses, was recovered comparably with PEG + TRIzol. This indicated that the nonenveloped surrogate (MS2) did not necessarily validate the efficient recovery for enveloped viruses. EMV + QIAamp Viral RNA Mini Kit provided significantly different φ6 recovery (1.6–21%) among the types of raw sewage. Then, the applicability of modified PEG + TRIzol was examined for the raw sewage collected in Tokyo, Japan. Of the 12 grab samples, 4 were positive for SARS-CoV-2 CDC N1 and N3 assay. Consequently, PEG + TRIzol provided the highest φ6 recovery and allowed for the detection of SARS-CoV-2 RNA from raw sewage.

Effects of cerium and tungsten substitution on antiviral and antibacterial properties of lanthanum molybdate.

Powders of cerium (Ce)-substituted and tungsten (W)-substituted La2Mo2O9 (LMO) were prepared using polymerizable complex method. Their antiviral and antibacterial performances were then evaluated using bacteriophage Qβ, bacteriophage Φ6, Escherichia coli, and Staphylococcus aureus. The obtained powders, which were almost single-phase, exhibited both antiviral and antibacterial properties. Effects of dissolved ions on their antiviral activity against bacteriophage Qβ were remarkable. A certain contribution of direct contact to the powder surface was also inferred along with the dissolved ion effect for antiviral activity against bacteriophage Φ6. Dissolved ion effects and pH values suggest that both Mo and W are in the form of polyacids. Antiviral activity against bacteriophage Φ6 was improved by substituting Ce for La in LMO. Similarly to LMO, Ce-substituted LMO exhibited hydrophobicity. Inactivation of alkaline phosphatase enzyme proteins was inferred as one mechanism of the antiviral and antibacterial activities of the obtained powders.

(Invited) Single-Molecule Electronics for DNA Sequencing

Nanoscale materials provide new opportunities to interface solid-state electronics with biomolecules and biochemical activity. For example, single-walled carbon nanotubes (SWNTs) have the special property of electronic resistance that is sensitive to single electrons. We have exploited this sensitivity to build nanoelectronic biosensors that monitor the biochemical activity of individual proteins [1]. As an attached protein moves, binds, or performs catalysis, its charged amino acid sidechains induce resistance fluctuations in the SWNT device that may be monitored with microsecond resolution [2].Recently, we have used this measurement platform for single-molecule measurements of DNA polymerases [3]. Polymerases are the key enzymes for converting single-stranded DNA to double-stranded helices, the primary step in DNA replication, amplification, and most sequencing technologies. When a polymerase processing DNA is also attached to a SWNT device, the electrical signal provides a high-resolution readout of single-nucleotide incorporations and exciting possibilities for high-density, high-throughput electronic DNA sequencing.To investigate the feasibility of electronic DNA sequencing, we have compared single-molecule transduction by DNA polymerases from three different organisms. By working with multiple families of DNA polymerases, we have tested the applicability of the electronic technique while also generating detailed records of differences among the enzymes. For example, we observe an anomalous rate variability when measuring the polymerase from the bacillus phage φ29. Base incorporation rates average 20 s-1 for most the enzymes processing single-stranded DNA templates, but rates up to 200 and 400 s-1 occurred when φ29 encountered homopolymeric sequences of poly(dT) or poly(dC), respectively. When processing poly(dA) and poly(dG) sequences, on the other hand, φ29 had bursts of activity interrupted by pauses lasting 50 to 300 s. This sequence-dependent activity illustrates one way that single-molecule methods reveal information hidden in ensemble-based techniques.Another workhorse protein in DNA sequencing technologies is the DNA polymerase derived from the thermophilic bacteria Thermus aquaticus (Taq). Anomalous stability at high temperatures makes Taq a unique enzyme for the polymerase chain reaction (PCR) and commercial amplification of DNA. In a first for single-molecule biophysics, SWNT devices have recorded Taq activity over a wide temperature range from 22 to 94 °C, including the typical PCR operating temperature of 72 °C. Even operating at this high temperature, the technique resolved Taq testing incoming nucleotides for complementarity and incorporating correct matches in the base-by-base construction of Watson-Crick pairs. The detailed recordings reveal the similarities of Taq’s operation to other, room temperature polymerases.[1] Y. Choi, et. al., Science 335, 319 (2012). [2] M. V. Akhterov, et. al., ACS Chem. Biol. 10, 1495 (2015). [3] T. J. Olsen et. al., JACS 135, 7855 (2013); O. T. Gul et. al., Biosensors 6, 29 (2016)

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases.

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1–1000 s duration, of which the short-lived ones (1–10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

Use of phage ϕ6 to inactivate Pseudomonas syringae pv. actinidiae in kiwifruit plants: in vitro and ex vivo experiments

Over the last years, the global production and trade of kiwifruit has been severely impacted by Pseudomonas syringae pv. actinidiae (Psa), a phytopathogen that causes a disease in kiwifruit plants known as bacterial canker. The available treatments for this disease are still scarce, with the most common involving frequently spraying the orchards with disinfectants, copper-based bactericides and/or antibiotics. Moreover, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Phage therapy may be an alternative approach to inactivate Psa. The present study investigated the potential application of the already commercially available bacteriophage (or phage) ϕ6 to control Psa infections. The inactivation of Psa was assessed in vitro, using liquid culture medium, and ex vivo, using artificially contaminated kiwifruit leaves with two biovar 3 (a highly aggressive pathogen) strains (Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10). In the in vitro experiments, the phage ϕ6 was effective against both strains (maximum reduction of 2.2 and 1.9CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). In the ex vivo tests, the decrease was lower (maximum reduction 1.1 log and 1.8CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). The results of this study suggest that the commercially available phage ϕ6 can be an effective alternative to control Psa infections in kiwifruit orchards.

Efficiency of Phage φ6 for Biocontrol of Pseudomonas syringae pv. syringae: An in Vitro Preliminary Study.

Pseudomonas syringae is a plant-associated bacterial species that has been divided into more than 60 pathovars, with the Pseudomonas syringae pv. syringae being the main causative agent of diseases in a wide variety of fruit trees. The most common treatments for biocontrol of P. syringae pv. syringae infections has involved copper derivatives and/or antibiotics. However, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Therefore, it is essential to search for new approaches for controlling P. syringae pv. syringae. Phage therapy can be a useful alternative tool to the conventional treatments to control P. syringae pv. syringae infections in plants. In the present study, the efficacy of bacteriophage (or phage) φ6 (a commercially available phage) was evaluated in the control of P. syringae pv. syringae. As the plants are exposed to the natural variability of physical and chemical parameters, the influence of pH, temperature, solar radiation and UV-B irradiation on phage φ6 viability was also evaluated in order to develop an effective phage therapy protocol. The host range analysis revealed that the phage, besides its host (P. syringae pv. syringae), also infects the Pseudomonas syringae pv. actinidiae CRA-FRU 12.54 and P. syringae pv. actinidiae CRA-FRU 14.10 strains, not infecting strains from the other tested species. Both multiplicities of infection (MOIs) tested, 1 and 100, were effective to inactivate the bacterium, but the MOI 1 (maximum reduction of 3.9 log CFU/mL) was more effective than MOI 100 (maximum reduction of 2.6 log CFU/mL). The viability of phage φ6 was mostly affected by exposure to UV-B irradiation (decrease of 7.3 log PFU/mL after 8 h), exposure to solar radiation (maximum reduction of 2.1 PFU/mL after 6 h), and high temperatures (decrease of 8.5 PFU/mL after 6 days at 37 °C, but a decrease of only 2.0 log PFU/mL after 67 days at 15 °C and 25 °C). The host range, high bacterial control and low rates of development of phage-resistant bacterial clones (1.20 × 10−3) suggest that this phage can be used to control P. syringae pv. syringae infections in plants, but also to control infections by P. syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit. Although the stability of phage φ6 was affected by UV-B and solar radiation, this can be overcome by the application of phage suspensions at the end of the day or at night.

Preparation of hydrophobic La2Mo2O9 ceramics with antibacterial and antiviral properties.

After powder of La2Mo2O9 (LMO) was prepared using complex polymerization, dense sintered bodies (96% relative density) of LMO were obtained from the powder through pressureless sintering in a synthesized air atmosphere. The water contact angle of the LMO ceramics increased gradually during storage in ambient air. It reached 93.6 ± 3.0° in 624 h. Results of XPS analysis and ozone treatment suggest that organic substances in ambient air adsorbed onto the LMO surface during storage. Measurements of antibacterial (Escherichia coli and Staphylococcus aureus) and antiviral (bacteriophage Qβ and bacteriophage Φ6) activities of LMO revealed that their survival rates decreased more than 99.9% within 6 h. Based on results obtained using dissolved ion contact method and from comparison of the antibacterial and antiviral activities with La2O3 and MoO3, one can infer that the synergistic effect of La2O3 and MoO3 plays an important role in the high antibacterial and antiviral activity of LMO.

Structural assembly of the tailed bacteriophage \u03d529

The mature virion of the tailed bacteriophage ϕ29 is an ~33 MDa complex that contains more than 450 subunits of seven structural proteins assembling into a prolate head and a short non-contractile tail. Here, we report the near-atomic structures of the ϕ29 pre-genome packaging head (prohead), the mature virion and the genome-emptied virion. Structural comparisons suggest local rotation or oscillation of the head-tail connector upon DNA packaging and release. Termination of the DNA packaging occurs through pressure-dependent correlative positional and conformational changes in the connector. The funnel-shaped tail lower collar attaches the expanded narrow end of the connector and has a 180-Å long, 24-strand β barrel narrow stem tube that undergoes conformational changes upon genome release. The appendages form an interlocked assembly attaching the tail around the collar. The membrane active long loops at the distal end of the tail knob exit during the late stage of infection and form the cone-shaped tip of a largely hydrophobic helix barrel, prepared for membrane penetration.