Detection of nucleic acid lesions during photochemical inactivation of RNA viruses by treatment with methylene blue and light using real-time PCR

The mechanism of bacteriophage photoinactivation by methylene blue and light (MB+L) involves genomic RNA damage. In this study, two RNA viruses, Sindbis virus (SINV) and hepatitis C virus were treated by MB+L and their nucleic acids were amplified to show that RNA lesions occurred during inactivation. During MB+L inactivation, the viral load of both viruses was significantly reduced as MB+L exposure increased. The nucleic acid amplification of treated viral RNA was inhibited in a time-dependent manner and the percentage inhibition of amplification reached about 99% after 30 min of treatment. Furthermore, as compared to SINV viral infectivity detected by quantification of the 50% tissue culture infective dose (TCID(50)), the inhibition of SINV RNA amplification strongly correlated with a decrease in in vitro infectivity (R(2) > 0.94), suggesting that RNA serves as the main target during MB+L inactivation.     

Strategies for evaluation of enveloped virus inactivation in red cell concentrates using hypericin

Photodynamically induced virus inactivation appears promising in preventing transmission of enveloped virus infections in transfusible blood products. The potential for utilizing hypericin as a photosensitizer to inactivate key enveloped viruses in packed red cell concentrates (PRC) was evaluated. In addition to inactivating effectively > or = 10(6) TCID50 of human immunodeficiency virus (HIV), inactivation of bovine viral diarrhea virus (BVDV) in PRC was used as a model for hepatitis C virus to overcome the deficiency in reliable experimental systems for hepatitis C virus (HCV) inactivation. BVDV was two orders of magnitude more sensitive to inactivation by hypericin than HIV. As part of the virucidal efficacy analyses, the effects of photosensitization on hemopoietic cell lines carrying quiescent integrated HIV provirus were studied as models for evaluating virus inactivation in latently infected cells. Phorbol ester-induced virus production by these cells was effectively prevented by photosensitization with hypericin. A refinement of the illumination conditions, incorporating a monochromatic sodium light source with an emission spectrum coinciding with the absorption peak of hypericin, was highly virucidal, however, caused unacceptable levels of hemolysis. Red blood cells could be protected from phototoxic cellular damage by complexing hypericin with human serum albumin (albumin-hypericin), but the decrease in hemolysis was at the expense of virucidal efficacy. Thus, excitation of hypericin with a fluorescent source appears to be useful potentially for virus inactivation in PRC.     

Virus photoinactivation in stroma-free hemoglobin with methylene blue or 1,9-dimethylmethylene blue

Photoinactivation of vesicular stomatitis virus (VSV) in stroma-free hemoglobin (SFH) was carried out using methylene blue (MB) or 1,9-dimethylmethylene blue (DMMB). The VSV was more sensitive to inactivation by 660 nm light with 1 microM DMMB than with the same concentration of MB. Under conditions that inactivated 6 log10 of VSV, the methemoglobin content (Met-Hb[%]) and P50 of hemoglobin were changed by 1 microM MB phototreatment but were not changed by 1 microM DMMB phototreatment. The migration of hemoglobin during electrophoresis and the activity of superoxide dismutase were not changed by MB or DMMB phototreatment. In contrast to the results obtained with DMMB at 660 nm, 580 nm irradiation of SFH with DMMB resulted in a significant increase of Met-Hb(%) under conditions that only inactivated 1.19 log10 VSV. The 580 nm irradiation primarily activates the dimer and higher-order aggregates of the dyes, while 660 nm irradiation primarily activates the monomer. These results indicate that the monomer form of DMMB can effectively inactivate viruses without damage to SFH.     

Factors Affecting Virus Photoinactivation by a Series of Phenothiazine Dyes

A series of four phenothiazine dyes, including methylene blue (MB), were previously tested for their ability to photoinactivate viruses in red cell suspensions. One of the dyes, 1,9-dimethyl-3-dimethylamino-7-dimethylaminophenothiazine (1,9-dimethylmethylene blue), exhibited good intracellular and extracellular virucidal activity for several RNA and DNA viruses under conditions that minimally affected red cell properties. In order to understand why the virucidal specificity of 1, 9-dhnethylmethylene blue was greater than other phenothiazines tested, the physical and chemical properties of the dye were compared to three other closely related analogues (MB, 1,9-dimethyl-3-diethylamino-7-dlbutylaminophenothiazine [compound 4-140], 1,9-dimethyl-3-dimethylamino-7-diethylaminophenothiazine [compound 6-136]). All compounds required light and oxygen for virucidal activity and had relatively high singlet oxygen yields (>0.5), but 1,9-dimethylmethylene blue had a singlet oxygen yield approximately 50% greater than that of MB. In addition, the hydrophobicity/hydophilicity of the compounds varied, with the partition coefficients (2-octanol : water) ranging from 0.11 for MB to 3560 for compound 4-140. The dyes had the following affinities for DNA: 1,9-dimethylmethylene blue > compound 6-136 > MB ～ compound 4-140. This order was similar to the order of activities for photoinactivation of the nonenveloped bacteriophage, R17, by the four compounds. Results with the most hydrophobic compound, 4-140, contrasted with those obtained with 1,9-dimethylmethylene blue. Compound 4-140 had a high affinity for protein and a low affinity for DNA. Although compound 4-140 and light inactivated the nonenveloped bacteriophage R17 poorly, the dye readily photoinactivated enveloped viruses in buffer. However, unlike results with 1,9-dimethylmethylene blue, viral inactivation of enveloped viruses by compound 4-140 was completely inhibited by the presence of red cells and plasma. Thus, the high affinity of 1,9-di-methyymethylene blue for DNA and the dye's efficient singlet oxygen yield suggest viral nucleic acid as a potential target, which could explain the photosensitizer's ability to inactivate viruses without adversely affecting anuclete red cells.     

Simultaneous determination of mebeverine hydrochloride and sulpiride using the first derivatives of ratio spectra and chemometric methods.

Two methods are described for the simultaneous determination of mebeverine hydrochloride (MB) and sulpiride (SU) in their combinations. The first method depends on the first derivative of the ratio spectra by measurement of the amplitudes at 263.7 and 234.9 nm for MB and SU, respectively. The linear ranges and detection limits are 4.0-40.0 and 0.72 microg/ml for MB and 1.0-10.0 and 0.34 microg/ml for SU. In the second case, a chemometric (classical least squares) method was developed. The concentration data matrices were obtained by using different concentrations of pure drugs in 0.1 M HCl. The absorbance data matrix corresponding to each concentration data matrix was obtained by the measurements of absorbances in the range 200-300 nm in their zero order spectra; then calibration was obtained by using the absorbance data matrix and the concentration data matrix for the prediction of the unknown concentrations of MB and SU in their mixture. The numerical values were calculated by using Matlab R12 version 6.0 and Origin 5.0 software. The procedures do not require any separation steps. These two methods were successfully applied for assaying the pharmaceutical formulation, of Colona tablets.     

ANTIVIRAL ACTIVITY OF GILVOCARCIN V PLUS UVA RADIATION

Abstract Gilvocarcin V (GV), a coumarin, is a nucleic acid photosensitizer that is phototoxic to bacteria and mammalian cells at picomolar levels in the presence of near-UV radiation (UVA). We evaluated the effectiveness of GV plus UVA for inactivation of several viruses, including herpes simplex virus, type 1 (HSV) and the bacterial viruses φX174, T7, PRD1 and φ6. Some inactivation of the bacterial viruses was observed with UVA radiation alone (4–50% survival at 26 kJ/m²). Additional photosensitized inactivation was observed only with T7 and φ6 at 2.0 μ M GV. On the other hand, HSV was photoinactivated with concentrations of GV three orders of magnitude lower (1.0 n M ). Similar to the case with UV (254 nm) inactivation, the GV-UVA survival curve for HSV indicated multicomponent inactivation kinetics, which could not be explained by photobleaching of GV. The wide range of photosensitivities of these viruses to GV cannot be adequately explained by models based only on viral nucleic acid content or presence of lipid envelopes.     

INACTIVATION OF LIPID-CONTAINING VIRUSES BY HYDROPHOBIC PHOTOSENSITIZERS AND NEAR-ULTRAVIOLET RADIATION

Abstract—The hydrophobic photosensitizers acridine and phenothiazine inactivate the lipid-contnining viruses PM2,φ6, and herpes simplex when samples are illuminated with near-UV radiation. φ23–1- a . which is insensitive to organic solvents and presumably contains no lipids. is not inactivated under comparable conditions. For acridinc, the inactivation of virus requires that oxygen be present and is inhibited by sodium azide, implicating the involvement of singlet oxygen. For phenothiazine, oxygen is not required for photosensitized inactivation. Treatment of PM2 with acridine and near-UV light caused a complete disruption of the virion, as determined by sucrose gradient analysis of treated and untreated samples. These data and related observations suggest that lipid-containing viruses are inactivated through photosensitized membrane damage.     

Characterization of the Effects of Aryl-azido Compounds and UVA Irradiation on the Viral Proteins and Infectivity of Human Immunodeficiency Virus Type 1

Hydrophobic UV-activatable compounds have been shown to partition into the hydrophobic region of biological membranes to selectively label transmembrane proteins, and to inactivate enveloped viruses. Here, we analyze various UV-activatable azido- and iodo-based hydrophobic compounds for their ability to inactivate a model-enveloped virus, human immunodeficiency virus (HIV-1 MN). Treatment of HIV-1 with 1,5-diazidonapthalene (DAN), 1-iodo, 5-azidonaphthalene (INA), 1-azidonaphthalene (AzNAP) or 4,4′-diazidobiphenyl (DABIPH) followed by UVA irradiation for 2 min resulted in complete viral inactivation, whereas treatment using analogous non–azido-containing controls had no effect. Incorporation of an azido moiety within these hydrophobic compounds to promote photoinduced covalent reactions with proteins was found to be the primary mechanism of viral inactivation for this class of compounds. Prolonged UVA irradiation of the virus in the presence of these azido compounds resulted in further modifications of viral proteins, due to the generation of reactive oxygen species, leading to aggregation as visualized via Western blot analysis, providing additional viral modifications that may inhibit viral infectivity. Furthermore, inactivation using these compounds resulted in the preservation of surface antigenic structures (recognized by neutralizing antibodies b12, 2g12 and 4e10), which is favorable for the creation of vaccines from these inactivated virus preparations.     

Purification and enrichment of virus samples utilizing magnetic beads on a microfluidic system

This study reports a new microfluidic system with three integrated functional devices for pumping, mixing and separation of bio-samples by utilizing micro-electro-mechanical-systems technology. By using antibody-conjugated magnetic beads, the developed system can be used to purify and enrich virus samples such that the subsequent detection of viruses can be performed with a higher sensitivity. The target viruses were first captured by the antibody coated onto the magnetic beads by using a rotary micromixer which performed the incubation process. The viruses were then purified and enriched by a magnetic field generated by planar microcoils. The integrated microfluidic system can perform the whole purification and enrichment process automatically using a rotary micropump and appropriate microvalves. In addition, a numerical simulation was also employed to optimize the design of the microcoils and to investigate the magnetic field strength and distribution. The simulation results were consistent with experimental observations. Finally, the developed system was used to successfully perform the purification and enrichment of Dengue viruses. The detectable limit of Dengue viruses was found to be as low as 10(2) pfu ml(-1) by using this approach. Therefore, the integrated microsystem can perform incubation, transportation, mixing and purification of virus samples, possibly making it a promising platform for future biological and medical applications.     

Photodynamic inactivation of retroviruses by phthalocyanines: the effects of sulphonation, metal ligand and fluoride.

The photodynamic inactivation of retroviruses was investigated using aluminium and zinc phthalocyanine (Pc) derivatives. The N2 retrovirus packaged in either of the two murine cell lines, Psi2 and PA317, was used as a model for enveloped viruses. AlPc derivatives were found to be more effective photodynamically for inactivation of the viruses than the corresponding ZnPc derivatives. Sulphonation of the Pc macrocycle reduced its photodynamic activity progressively for both AlPc and ZnPc. Fluoride at 5 mM during light exposure completely protected viruses against inactivation by AlPc. In the presence of F-, inactivation by the sulphonated derivatives AlPcS1 and AlPcS4 was reduced 2.5- and twofold respectively. In a biological membrane (erythrocyte ghosts), F- had no significant effect on AlPcS4-sensitized lipid peroxidation. Under similar conditions, cross-linking of spectrin monomers in ghosts is drastically inhibited (E. Ben-Hur and A. Orenstein, Int. J. Radiat. Biol., 60 (1991) 293-301). Since Pc derivatives do not inactivate non-enveloped viruses, it is hypothesized that inactivation occurs by photodynamic damage to envelope protein(s). Substitution of sulphonic acid residues reduces the binding of Pc derivatives to the envelope protein(s), thereby diminishing their photodynamic efficacy and the ability of F- to modify it.     

Photodynamic inactivation of methylene blue and tungsten-halogen lamp light against food pathogen Listeria monocytogenes

The aim of this study was to verify the bactericidal effect and the damage of photodynamic inactivation (PDI) using methylene blue (MB) and tungsten-halogen lamp over Listeria monocytogenes via atomic force microscopy, absorption spectrophotometry, agarose gel electrophoresis, real-time PCR and SDS-PAGE. The obtained data indicated that the viability of L. monocytogenes was ca 7-log reduced by illumination with 10 min tungsten-halogen lamp light under the presence of 0.5 μg mL(-1) MB, and this bactericidal activity against L. monocytogenes of PDI increased proportionally to the concentration of MB and the duration of irradiation. Moreover, after irradiation with MB and visible light, the leakage of intracellular contents was estimated by spectrophotometer at OD(260) and OD(280), which correlated with morphological alterations. Furthermore, genomic DNA cleavage and protein degradation were also detected after PDI treatment. Consequently, breakage of the membrane, damage of the genomic DNA and degradation of bacterial proteins may play an important role in the mechanisms involved in PDI-MB bactericidal activity on L. monocytogenes.     

Enhanced wavelength-sensitivity of volume holographic grating with dual-photoinitiation system in polymer-dispersed liquid crystal

We propose a method to improve the wavelength-sensitivity of a volume holographic grating by mixing dual-photoinitiation dopants, namely, rose bengal (RB)/N-phenylglycine (NPG) system and methylene blue (MB)/p-toluenesulfonic acid (PTSA) system, which enable the fabrication of gratings upon both green and red light illumination. The RB/NPG green light photoinitiation system is chosen in combination with the red-active MB/PTSA system because RB and MB absorb light near 567 and 665 nm independently. In this case, the holographic gratings, recorded in polymer-dispersed liquid crystal, can be fabricated simultaneously by two different visible laser lights with an output wavelength of 532 and 632.8 nm. The electro-optical performances of the gratings exposed upon 532 and 632.8 nm have been implemented: the diffraction efficiencies (DE) are 75% and 57% respectively when the exposure time is optimised to 2 min and 17 min; the threshold voltage is approximately 2.9 V/μm. The enhanced wavelength-sensitivity of holographic gratings allows for the storage of three-dimensional (3D) images on the same hologram plate, and these 3D images are easily reconstructed by both red and green light.     

IMPORTANCE OF TYPE I AND TYPE II MECHANISMS IN THE PHOTODYNAMIC INACTIVATION OF VIRUSES IN BLOOD WITH ALUMINUM PHTHALOCYANINE DERIVATIVES

The relative importance of type I and type II mechanisms in the photodynamic treatment of red blood cell concentrations (RBCC) to inactivate viruses was studied using aluminum phthalocyanine tetrasulfonate (AlPcS4), visible light and quenching or enhancing agents of reactive forms of oxygen. Treatment of a human RBCC with 10-13 microM AlPcS4 and 25-26 mW/cm2 visible light resulted in the rapid and complete inactivation of added vesicular stomatitis virus (VSV). The addition of mannitol, glycerol, reduced glutathione (GSH), or superoxide dismutase (SOD), known quenching agents of type I mechanisms, had little to no effect on the rate of inactivation of VSV. Significant inhibition of VSV kill was observed on addition of tryptophan or sodium azide, known quenchers of type II mechanisms. Additionally, the rate of VSV kill was enhanced in the presence of D2O. Taken together, these results indicate a predominant role of singlet oxygen in the inactivation of VSV on photodynamic treatment of RBCC. The relative importance of type I and type II mechanisms on cellular toxicity was also evaluated. Little, if any hemoglobin release was observed on treatment of human or rabbit RBCC with 10 microM AlPcS4 and 44 J/cm2 of visible light in the presence or absence of the above mentioned quenchers. The effect of the addition of quenchers on the recovery and circulatory survival of treated, autologous rabbit RBCC, labeled with 51Cr, was also assessed.(ABSTRACT TRUNCATED AT 250 WORDS)     

PHOTOREACTIVATION OF DNA-CONTAINING CAULIFLOWER MOSAIC VIRUS AND TOBACCO MOSAIC VIRUS RNA ON DATURA

Abstract— Datura stramonium L. is a local lesion host for TMV-RNA and DNA-containing cauliflower mosaic virus (CAMV). Datura can photorepair UV-damaged TMV-RNA and CAMV, giving photoreac-tivation sectors of 0.40 and 0. 33 , respectively. Dose response curves for photoreactivation of TMV-RNA and CAMV show that 4540 min of cool white light (15 W.m^-2) is required for maximum photoreactivation. Blue light and near UV are equally effective in photoreactivating UV-irradiated TMV-RNA, whereas near UV is initially more effective than blue light for the photorepair of UV-inactivated CAMV. Higher doses of near UV apparently inactivate the CAMV photorepair system. In the case of CAMV, photoreactivating light must be applied immediately after inoculation with the virus. Two to three hours of incubation in the dark after inoculation results in complete loss of response to photoreactivating irradiation. In contrast, limited photoreactivation of TMV-RNA occurs even after 4 h of dark incubation after inoculation, although photoreactivating irradiation is most effective when applied immediately after inoculation. Light is required for the maintenance of photoreactivation for both TMV-RNA and CAMV. Daturas placed in the dark for six days lose their ability to photoreacti-vate. Recovery of the TMV-RNA photorepair system is rapid; complete recovery attained with 90 or more min of white light (15 W m-'). Recovery of CAMV photorepair system is slow; 90% recovery attained after only 20 h of light. However, full recovery can be induced by as little as 6h of light when CAMV is inoculated 24 h after the onset of illumination. These results suggest two photorepair systems are present in Datura .     

APPLICATION OF PHOTODYNAMIC OXIDATION TO THE DISINFECTION OF TAPWATER, SEA WATER, AND SEWAGE CONTAMINATED WITH POLIOVIRUS

Abstract. Poliovirus when added to tapwater, sewage or seawater was readily photoinactivated by methylene blue and visible light. Typically, almost 2.5 logs of virus could be inactivated upon a 5-min exposure to 670 nm light (20 W/m²) in solutions containing 13 μ M methylene blue at pH 10.0. A biphasic inactivation curve was produced for poliovirus, regardless of dye concentration, pH, temperature, sensitization time, nature of suspending solution or sequence of light exposure. These results indicated that a multi-hit inactivation event was occurring. Preincubation of the dye-virus mixture at 24°C increased the rate of virus photoinactivation. Dye concentrations above 26 μ M have little advantage in increasing the amount of virus photoinactivated. Significant inactivation of the virus in the dark occurred at high dye concentrations (52–130μ M ).     

THE ROLE OF DNA DAMAGE IN PM2 VIRAL INACTIVATION BY METHYLENE BLUE PHOTOSENSITIZATION

Abstract— This study investigates the importance of DNA damage in viral inactivation by phenothiazines and light. Phenothiazines, including methylene blue (MB), toluidine blue and azure B are of particular interest because of their ability to bind to nucleic acids in vitro. Initial studies employing phages T7, MS2 and PM2 indicated that both DNA and RNA phages as well as enveloped and nonenveloped phages can be inactivated by phenothiazine photosensiti-zation. PM2, which contains a lipid-protein bilayer and supercoiled DNA, was used for the mechanistic studies to model blood-borne viruses. Viral DNA damage was assessed following treatment of phage to known levels of viral inactivation by extracting the DNA and analyzing for both direct and piperidine-catalyzed strand cleavage by gel electrophoresis. DNA strand cleavage was found to be both sensitizer concentration and light dose dependent. Both viral inactivation and DNA damage were found to be oxygen-dependent events. In parallel experiments, strand cleavage of isolated PM2 DNA treated with MB and light was also found to be oxygen dependent, in contrast to some previous reports. Transfection studies, which measure the infectivity of the extracted viral DNA, indicated that DNA from MB-treated phage was just as capable of generating progeny virus as the untreated controls. It was therefore concluded that the observed DNA damage is not correlated with loss of phage infectivity.     

Effect of the TAT-RasGAP(317-326) peptide on apoptosis of human malignant mesothelioma cells and fibroblasts exposed to meso-tetra-hydroxyphenyl-chlorin and light

BACKGROUND: 5,10,15,20-Tetrakis(m-hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy (PDT) has shown insufficient tumor selectivity for the treatment of pleural mesothelioma. Tumor selectivity of mTHPC-PDT may be enhanced in the presence of the TAT-RasGAP(317-326) peptide which has the potential to specifically sensitize tumor cells to cytostatic agents. MATERIALS AND METHODS: H-meso-1 and human fibroblast cell cultures, respectively, were exposed to two different mTHPC doses followed by light delivery with and without TAT-RasGAP(317-326) administration. mTHPC was added to the cultures at a concentration of 0.04microg/ml and 0.10microg/ml, respectively, 24h before laser light illumination at 652nm (3J/cm(2), 40mW/cm(2)). TAT-RasGAP(317-326) was added to the cultures immediately after light delivery at a concentration of 20microM. The apoptosis rate was determined by scoring the cells displaying pycnotic nuclei. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Light delivery associated with 0.04microg/ml mTHPC resulted in a significantly higher apoptosis rate in the presence of TAT-RasGAP(317-326) than without in H-meso-1 cells (p<0.05) but not in fibroblasts. In contrast, 1.0microg/ml mTHPC and light resulted in a significantly higher apoptosis rate in both H-meso-1 cells and fibroblasts as compared to controls (p<0.05) but the addition of TAT-RasGAP(317-326) did not lead to a further significant increase of the apoptosis rate of both H-meso-1 cells and fibroblasts as compared to mTHPC and light delivery alone. CONCLUSION: TAT-RasGAP(317-326) selectively enhanced the effect of mTHPC and light delivery on H-meso-1 cells but not on fibroblasts. However, this effect was mTHPC dose-dependent and occurred only at a low sensitizer dose.     

Identification of novel target sites and an inhibitor of the dengue virus E protein

Dengue and related flaviviruses represent a significant global health threat. The envelope glycoprotein E mediates virus attachment to a host cell and the subsequent fusion of viral and host cell membranes. The fusion process is driven by conformational changes in the E protein and is an essential step in the virus life cycle. In this study, we analyzed the pre-fusion and post-fusion structures of the dengue virus E protein to identify potential novel sites that could bind small molecules, which could interfere with the conformational transitions that mediate the fusion process. We used an in silico virtual screening approach combining three different docking algorithms (DOCK, GOLD and FlexX) to identify compounds that are likely to bind to these sites. Seven structurally diverse molecules were selected to test experimentally for inhibition of dengue virus propagation. The best compound showed an IC₅₀ in the micromolar range against dengue virus type 2. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Protein/Peptide Direct Virus Inactivation During Chromatographic Process: Developing Approaches

Virus clearance is required for pharmaceutical preparations derived from animal or human sources such as blood products, vaccines, recombinant proteins produced in mammalian cell lines, etc. High cost and substantial protein losses during virus inactivation are significant problems for protein/peptide manufacturing. The goal of this project was to develop a method to perform virus inactivation in a course of protein chromatographic purification. Another goal was to show that the chromatographic adsorbent can serve as reliable “sieva” for mechanical washing away of infecting viruses. Using chromatographic, photometric, IFA, and RT-PCR approaches, it was discovered that high temperature-depending dynamic capacity of adsorbent allowed to perform a virus inactivation directly in a chromatographic column by solvent/detergent treatment. The peptide/protein biological activity was completely preserved. Using this new approach enveloped and nonenveloped viruses were effectively removed protein preparation. In addition, it was shown that RT-PCR method demonstrates more precise and reproducible results and robust properties for assessment of virus reduction than virus titer followed by infectivity studies. Presented method allowed to obtain the factor of virus concentration decrease (FVD) values that were higher than those provided by known technologies and was sufficient for a full inactivation of viruses. The method is recommended to use in pharmaceutical industry.