Role of Phage Capsid in the Resistance to UV-C Radiations.

The conformational variation of the viral capsid structure plays an essential role both for the environmental resistance and acid nuclear release during cellular infection. The aim of this study was to evaluate how capsid rearrangement in engineered phages of M13 protects viral DNA and peptide bonds from damage induced by UV-C radiation. From in silico 3D modelling analysis, two M13 engineered phage clones, namely P9b and 12III1, were chosen for (i) chemical features of amino acids sequences, (ii) rearrangements in the secondary structure of their pVIII proteins and (iii) in turn the interactions involved in phage capsid. Then, their resistance to UV-C radiation and hydrogen peroxide (H2O2) was compared to M13 wild-type vector (pC89) without peptide insert. Results showed that both the phage clones acquired an advantage against direct radiation damage, due to a reorganization of interactions in the capsid for an increase of H-bond and steric interactions. However, only P9b had an increase in resistance against H2O2. These results could help to understand the molecular mechanisms involved in the stability of new virus variants, also providing quick and necessary information to develop effective protocols in the virus inactivation for human activities, such as safety foods and animal-derived materials.

Visible-light-driven photocatalytic inactivation of bacteriophage f2 by Cu-TiO2 nanofibers in the presence of humic acid.

Pathogenic viruses in drinking water are great threats to public health. Visible-light-driven photocatalysis is a promising technology for virus inactivation. However, the existing photocatalytic antiviral research studies have mostly been carried out in single-component systems, neglecting the effect of natural organic matter, which exists widely in actual water bodies. In this paper, electrospun Cu-TiO2 nanofibers were prepared as photocatalysts, and their photocatalytic antiviral performance in the presence of humic acid (HA) was comprehensively studied for the first time. The properties of the reaction mixture were measured during the reaction. In addition, the safety, reliability and stability of photocatalytic disinfection in the mixed system were evaluated. The results showed that the virus removal efficiency decreased with the increase of the HA concentration. The type of reaction solution, such as PBS buffer solution or water, did not affect the removal efficiency noticeably. Under acidic conditions, the electrostatic forces between photocatalysts and viruses were strengthened, leading to higher virus removal efficiency. As the reaction time went on, the pH value in the solution increased first and then tended to be stable, the conductivity remained stable, and the dissolved oxygen increased first and then decreased. The safety test showed that the concentration of Cu ions released into the solution was lower than specified by the international standards. No photoreactivation was observed, and the addition of HA significantly reduced the reutilization efficiency of the photocatalysts.

Dual UV irradiation-based metal oxide nanoparticles for enhanced antimicrobial activity in Escherichia coli and M13 bacteriophage.

Metal oxide (MO) nanoparticles have been studied as nano-antibiotics due to their antimicrobial activities even in antibiotic-resistant microorganisms. We hypothesized that a hybrid system of dual UV irradiation and MO nanoparticles would have enhanced antimicrobial activities compared with UV or MO nanoparticles alone. In this study, nanoparticles of ZnO, ZnTiO3, MgO, and CuO were selected as model nanoparticles. A dual UV collimated beam device of UV-A and UV-C was developed depending upon the lamp divided by coating. Physicochemical properties of MO nanoparticles were determined using powder X-ray diffractometry (PXRD), Brunauer-Emmett-Teller analysis, and field emission-scanning electron microscopy with energy-dispersive X-ray spectroscopy. Atomic force microscopy with an electrostatic force microscopy mode was used to confirm the surface topology and electrostatic characteristics after dual UV irradiation. For antimicrobial activity test, MO nanoparticles under dual UV irradiation were applied to Escherichia coli and M13 bacteriophage (phage). The UV-A and UV-C showed differential intensities in the coated and uncoated areas (UV-A, coated = uncoated; UV-C, coated ≪ uncoated). MO nanoparticles showed sharp peaks in PXRD patterns, matched to pure materials. Their primary particle sizes were less than 100 nm with irregular shapes, which had an 8.6~25.6 m2/g of specific surface area with mesopores of 22~262 nm. The electrostatic properties of MO nanoparticles were modulated after UV irradiation. ZnO, MgO, and CuO nanoparticles, except ZnTiO3 nanoparticles, showed antibacterial effects on E. coli. Antimicrobial effects on E. coli and phages were also enhanced after cyclic exposure of dual UV and MO nanoparticle treatment using the uncoated area, except ZnO nanoparticles. Our results demonstrate that dual UV-MO nanoparticle hybrid system has a potential for disinfection. We anticipate that it can be developed as a next-generation disinfection system in pharmaceutical industries and water purification systems.

Mutation spectrum resulting in M13mp2 phage DNA exposed to N-nitrosoproline with UVA irradiation.

N-nitrosoproline (NPRO) is endogenously formed from proline and nitrite. In an effort to delineate the mechanism of NPRO-induced photomutagenicity, we investigated the mutagenic spectrum of NPRO on M13mp2 DNA with UVA irradiation. Following exposure to NPRO and UVA, the mutation frequency increased significantly in an NPRO and UVA dose-dependent manner. The sequence data derived from seventy of the mutants indicated that mutagenesis resulted mainly from an increase in single-base substitutions, the most frequent being GC to CG transversions. Non-clustering of the GC to CG mutations suggests that NPRO+UVA damage to DNA is random. These transversions may be caused by guanine adducts in DNA or in part by oxidatively modified guanine in DNA exposed to NPRO and UVA.

No effect of femtosecond laser pulses on M13, E. coli, DNA, or protein.

Data showing what appears to be nonthermal inactivation of M13 bacteriophage (M13), Tobacco mosaic virus, Escherichia coli (E. coli), and Jurkatt T-cells following exposure to 80-fs pulses of laser radiation have been published. Interest in the mechanism led to attempts to reproduce the results for M13 and E. coli. Bacteriophage plaque-forming and bacteria colony-forming assays showed no inactivation of the microorganisms; therefore, model systems were used to see what, if any, damage might be occurring to biologically important molecules. Purified plasmid DNA (pUC19) and bovine serum albumin were exposed to and analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively, and no effect was found. DNA and coat proteins extracted from laser-exposed M13 and analyzed by AGE or PAGE found no effect. Raman scattering by M13 in phosphate buffered saline was measured to determine if there was any physical interaction between M13 and femtosecond laser pulses, and none was found. Positive controls for the endpoints measured produced the expected results with the relevant assays. Using the published methods, we were unable to reproduce the inactivation results or to show any interaction between ultrashort laser pulses and buffer/water, DNA, protein, M13 bacteriophage, or E. coli.

Studies of inactivation of encephalomyocarditis virus, M13 bacteriophage, and Salmonella typhimurium by using a visible femtosecond laser: insight into the possible inactivation mechanisms.

We report experimental results on the inactivation of encephalomyocarditis virus, M13 bacteriophage, and Salmonella typhimurium by a visible femtosecond laser. Our results suggest that inactivation of virus and bacterium by a visible femtosecond laser involves completely different mechanisms. Inactivation of viruses by a visible femtosecond laser involves the breaking of hydrogen∕hydrophobic bonds or the separation of the weak protein links in the protein shell of a viral particle. In contrast, inactivation of bacteria is related to the damage of their DNAs due to irradiation of a visible femtosecond laser. Possible mechanisms for the inactivation of viruses and bacteria are discussed.

Photonic approach to the selective inactivation of viruses with a near-infrared subpicosecond fiber laser.

We report a photonic approach for selective inactivation of viruses with a near-infrared subpicosecond laser. We demonstrate that this method can selectively inactivate viral particles ranging from nonpathogenic viruses such as the M13 bacteriophage and the tobacco mosaic virus to pathogenic viruses such as the human papillomavirus and the human immunodeficiency virus (HIV). At the same time, sensitive materials such as human Jurkat T cells, human red blood cells, and mouse dendritic cells remain unharmed. The laser technology targets the global mechanical properties of the viral protein shell, making it relatively insensitive to the local genetic mutation in the target viruses. As a result, the approach can inactivate both the wild and mutated strains of viruses. This intriguing advantage is particularly important in the treatment of diseases involving rapidly mutating viral species such as HIV. Our photonic approach could be used for the disinfection of viral pathogens in blood products and for the treatment of blood-borne viral diseases in the clinic.

Inactivation of viruses by coherent excitations with a low power visible femtosecond laser.

BACKGROUND: Resonant microwave absorption has been proposed in the literature to excite the vibrational states of microorganisms in an attempt to destroy them. But it is extremely difficult to transfer microwave excitation energy to the vibrational energy of microorganisms due to severe absorption of water in this spectral range. We demonstrate for the first time that, by using a visible femtosecond laser, it is effective to inactivate viruses such as bacteriophage M13 through impulsive stimulated Raman scattering. RESULTS AND DISCUSSION: By using a very low power (as low as 0.5 nj/pulse) visible femtosecond laser having a wavelength of 425 nm and a pulse width of 100 fs, we show that M13 phages were inactivated when the laser power density was greater than or equal to 50 MW/cm2. The inactivation of M13 phages was determined by plaque counts and had been found to depend on the pulse width as well as power density of the excitation laser. CONCLUSION: Our experimental findings lay down the foundation for an innovative new strategy of using a very low power visible femtosecond laser to selectively inactivate viruses and other microorganisms while leaving sensitive materials unharmed by manipulating and controlling with the femtosecond laser system.

Effect of synthetic hydroxy isothiocyanates on a bacterial virus and DNA.

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on psiK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.

Inactivation of viruses by laser-driven coherent excitations via impulsive stimulated Raman scattering process.

The inactivation of viruses such as M13 bacteriophages subject to excitations by a very low power visible femtosecond laser has been studied. Our experimental results show that for a visible femtosecond laser having lambda = 425 nm and a pulse width of 100 fs, the M13 bacteriophages are inactivated when the laser power density is greater than or equal to 49 MW/cm(2). The medium lethal laser power density (LD(50)) is 51.94+/-0.14 MW/cm(2). The functionality of M13 bacteriophages has been shown to be critically dependent on the pulse width as well as power density of the excitation laser. Our work demonstrates that by using a very low power visible femtosecond laser, it is plausible to inactivate viruses such as the M13 bacteriophages through impulsive stimulated Raman scattering process. These experimental findings suggest a novel avenue of selectively inactivating microorganisms while leaving the sensitive materials unharmed by manipulating and controlling with femtosecond laser systems.

Pyrimidine dimer formation and oxidative damage in M13 bacteriophage inactivation by ultraviolet C irradiation.

The mechanism by which UV-C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South-Western blotting for pyrimidine dimers. The involvement of singlet oxygen (1O2) was also investigated using azide and deuterium oxide and under deoxygenated conditions. With a decrease in M13 infectivity on irradiation, single-stranded circular genomic DNA (sc-DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc-DNA and linear-form DNA. However, the amount of sc-DNA remaining was not correlated with the survival of M13. The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PP) increased as a function of irradiation dose. The decrease in M13 infectivity was highly correlated with the increase in CPD and (6-4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 8-Oxo-7,8-dihydro-2'-deoxyguanosine did not form in the M13 genome after UV-C irradiation. Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide. Deoxygenation of the M13 suspension did not affect the inactivation, indicating that 1O2 did not participate in the inactivation of M13 by UV-C irradiation under these conditions. These results indicated that UV-C irradiation induced not only CPD and (6-4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity. The indirect effect of UV-C irradiation such as 1O2 production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV-C.

The influence of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZalpha gene of m13mp10.

One of the most predominating oxidative DNA damages, both spontaneously formed and after gamma-radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This 8oxoG is a mutagenic lesion because it can mispair with adenine instead of the correct cytosine leading to G:C to T:A transversions. In Escherichia coli (E. Coli) base excision repair (BER) is one of the most important repair systems for the repair of 8oxoG and other oxidative DNA damage. An important part of BER in E. coli is the so-called GO system which consists of three repair enzymes, MutM (Fpg), MutY and MutT which are all involved in repair of 8oxoG or 8oxoG mispairs. The aim of this study is to determine the effect of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZalpha gene. For that purpose, non-irradiated or gamma-irradiated double-stranded (ds) M13mp10 DNA, with the lacZalpha gene inserted as mutational target sequence was transfected into an E. coli strain which is deficient in both Fpg and MutY (BH1040). The resulting mutation spectra were compared with the mutation spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105) which were determined in an earlier study. The results of the present study indicate that combined Fpg- and MutY-deficiency induces a large increase in G:C to T:A transversions in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)) as compared to the fpg(-) and the wild type strain. Besides the increased levels of G:C to T:A transversions, there is also an increase in G:C to C:G transversions and frameshift mutations in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)).

DNA lesion and mutagenesis induced in phageM13mp2 by UVA, UVB and UVC irradiation.

Sunlight is carcinogenic and mutagenic and its genotoxic effects are believed to be the result of UV light-induced lesions in DNA. These lesions include pyrimidine dimers and (6-4) photoproducts, but it is uncertain whether the pyrimidine modifications are the sole pre-mutagenic lesions induced by UV light. Previous studies indicate that some sunlight-induced mutations in the single-stranded DNA phage M13mp2 may not be caused by these photoproducts. In this work, purified single-stranded phage DNA was exposed to UVA, UVB and UVC and the induced mutations were analyzed. All 3 types of UV light increase the mutation frequency. The mutants were sequenced and the results suggest that UVA exposure may induce formation of a non-dipyrimidine lesion in DNA.

Virus inactivation in superoxide dismutase preparations by ultraviolet light irradiation.

Viral inactivation in superoxide dismutase (SOD) derived from human red cells was carried out by ultraviolet light C (UVC) irradiation. With 400 J/m2 UVC irradiation, the titer of canine parvovirus (CPV, a nonenveloped virus), M13 bacteriophage (M13, a nonenveloped phage) and vesicular stomatitis virus (VSV, an enveloped virus), which were spiked into SOD solution, were reduced by > 4.6 log10 (detection limit), 7.0 log10 and 6.2 log10, respectively. The SOD activity was maintained and the band pattern of SOD on an electrophoresis gel was not changed even by 1000 J/m2 UVC irradiation. These results indicate that UVC irradiation is a promising method for the inactivation of both enveloped and nonenveloped viruses in SOD preparations while maintaining its activity.

Factors affecting M13 bacteriophage inactivation by methylene blue photosensitization.

We have investigated the factors that affect the virucidal activity of methylene blue (MB) photosensitization. The M13 bacteriophage was more rapidly inactivated at higher temperatures (6 degrees C < 24 degrees C < 38 degrees C). Rate constants for inactivation were 0.072, 0.139 and 0.260 (log10 inactivation)/ (J/cm2) at 6 degrees C, 24 degrees C and 38 degrees C, respectively. On the other hand, dye penetration into virus particles, which was monitored by the fluorescence of YOYO-1, was unchanged with incubation temperature. These data suggest that temperature dependency of M13 inactivation was due to factors other than dye permeability. The pH of the virus suspension also affected the rate of M13 inactivation by MB. The M13 bacteriophage was inactivated faster in basic suspensions and slower in acidic suspensions compared with neutral buffers. These results suggest that temperature and pH are factors that influence the extent of MB photosensitization, and hence, the control of these factors will be necessary for MB phototreatment of plasma products in transfusion medicine.

Mutations induced by gamma-irradiation of M13 bacteriophages containing single-stranded DNA.

Oxygenated suspensions of M13 bacteriophages, containing single-stranded M13mp10 DNA, were gamma-irradiated followed by infection of E. coli cells. Mutants in the mutational target sequence, which consists of the lac promoter /operator region, the lacZ alpha gene, and a 144 bp inframe insert in the lacZ alpha gene, were selected and characterized. Except for three one-base deletions, all of the 51 mutations characterized were base substitutions. All base substitutions appeared to involve guanines and cytosines and none affect adenines and thymines. Since most of the known repair systems do not act on single-stranded DNA, the conclusion can be drawn that radiation induces under these conditions only mutagenic damages on guanine and cytosine. Although all possible G- and C-transversions and transitions were found, there is a strong preference for G-->C and G-->T transversions (21 and 25% of all base substitutions, respectively) and C-->T transitions (48% of all base substitutions). These results indicate, that the G/C-->C/G and G/C-->T/A transversions, found after irradiation of double-stranded M13 DNA, are mainly due to radiation guanine products, whereas cytosine damage is mainly responsible for G/C-->A/T transitions.

Analysis of viral DNA, protein and envelope damage after methylene blue, phthalocyanine derivative or merocyanine 540 photosensitization.

Although numerous photosensitizers have been used experimentally to decontaminate viruses in cellular blood components, little is known about their mechanisms of photoinactivation. Using M13 bacteriophage and vesicular stomatitis virus (VSV) as model viruses, we have investigated alteration of the viral genome, protein and envelope after phototreatment. Methylene blue (MB) and aluminum phthalocyanine tetrasulfonate (AlPcS4) phototreatment inactivated bacteriophage M13 and decreased the fraction of single-stranded circular genomic DNA (sc-DNA) by converting it to linear form. This conversion was enhanced by treating the extracted DNA with piperidine at 55 degrees C. Piperidine-labile breaks were well correlated to phage survival (5.1% sc-DNA at 1.7% phage survival for MB) under conditions where only minor differences were seen in the relative abundance of M13 coat protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Neither aluminum phthalocyanine (AlPc) nor merocyanine 540 (MC540) inactivated M13 nor were there significant changes observed in DNA and coat protein. Methylene blue, AlPcS4 and AlPc inactivated VSV and inhibited fusion of the virus envelope to Vero cells at pH 5.7 (i.e. with plasma membrane). However, the degree of this inhibition was small compared to the extent of virus inactivation (43% inhibition vs. 4.7 log10 or 99.998% inactivation, for MB). In contrast, an antibody to VSV G-spike protein inhibited fusion at pH 5.7 by 52% with a concomitant decline in VSV infectivity of 0.15 log10 (30%). Few changes were observed in the relative abundance of G protein for MB and AlPcS4 phototreated samples and no additional protein bands were observed on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of thiols and oxygen on the survival of gamma-irradiated plasmid DNA and cells.

Some of the recent progress made in the understanding of the quantitative aspects of the oxygen effect in radiation biology by several groups is summarized. Examples are: the importance of unrepairable damage for the quantitative description of the oxygen effect; proof that protein thiols hardly contribute to protection in cells in the absence of oxygen; the proposal that protection by thiols in concentration ranges where all DNA radicals react with oxygen is due to the formation of hydroperoxides which can be repaired enzymatically by glutathione peroxydase; the finding that unscavengeable damage in plasmid DNA is mainly due to spur-induced clustered damages, but that the precursors of the scavengeable and the unscavengeable damage are comparably well repaired by thiols; the result that E. coli repair wild type strains are better protected by addition of thiols than strains with deficiencies in enzymatic repair capacities.

Quencher-enhanced specificity of psoralen-photosensitized virus inactivation in platelet concentrates.

BACKGROUND: Treatment with psoralens and UVA (PUVA) has been shown to be efficacious in eliminating the risk of virus transmission by platelet concentrates (PCs). It has previously been demonstrated that, during the inactivation of cell-free vesicular stomatitis virus (VSV) by aminomethyltrimethylpsoralen (AMT) and UVA in PCs, platelet function could be protected either by oxygen removal before irradiation or by inclusion of a type I free radical quencher, such as mannitol. STUDY DESIGN AND METHODS: Under previous PUVA treatment conditions for PCs (25 micrograms/mL AMT; 30 min UVA at 7 mW/cm2; 2 mM [2 mmol/L] mannitol), more than 6 log10 of added cell-free VSV was completely inactivated. In the current study, various PUVA conditions are evaluated for efficacy in inactivating other viral forms that could be present in PCs. Maintenance of platelet integrity (i.e., platelet number, solution pH, and aggregation response during initial storage after treatment) and kill of cell-associated VSV are examined. RESULTS: While cell-free viruses were inactivated efficiently under previous PUVA conditions, cell-associated VSV and the non-lipid-enveloped bacteriophage M13 were not. Effective inactivation of these viruses was achieved by raising the concentration of AMT to 50 micrograms per mL and extending the period of irradiation to 90 minutes (39 J/cm2). However, for maintenance of platelet integrity under these conditions, the prior removal of oxygen or the inclusion of compounds known to quench both type I and type II photoreactants (e.g., flavonoids such as rutin) was required. CONCLUSION: These findings suggest that the viral safety of PCs may be enhanced through treatment with AMT and UVA in the presence of flavonoids, and that flavonoid use may prove beneficial in other systems where oxygen-mediated damage occurs.