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.     

INVESTIGATIONS ON THE MECHANISM OF CHLORPROMAZINE PHOTOTOXICITY: EFFECTS ON LYSOSOMES OF CULTURED HUMAN FIBROBLASTS

Abstract The effect of chlorpromazine (CPZ) and UVA on lysosomes of cultured normal human fibroblasts has been investigated. Acid phosphatase (ACPase) activity in 12 000 g pellet of cells treated with CPZ (10 μg/m l ) and UVA (6 × 10⁴ J/m²) was found to be decreased as compared with non-treated, CPZ or UVA treated control cells. This decrease, however, was not accompanied by a concomitant increase in ACPase activity in the 12 000 g supernatant. The addition of Triton X-100 to cells pretreated with CPZ + UVA resulted in only a moderate increase in ACPase activity of the 12 000 g supernatant. ACPase activity of the cells incubated in media containing preirradiated CPZ was also found to he decreased. These results indicate that CPZ + UVA directly inactivate lysosomal enzymes, possibly without affecting the membrane.     

PHOTOINACTIVATION (365 NM) OF VACCINIA AND HERPES SIMPLEX VIRUSES INDUCED BY A NEW BUILT-IN DNA PHOTOSENSITIZER: 4-THIOTHYMIDINE

The thymidine analogue 4-thiothymidine (s⁴T) strongly absorbs light at wavelengths in the UVA range (Λ_max 335 nm) and we have examined the photoinactivation of vaccinia and herpes simplex viruses grown in the presence of this nucleoside. The cells used in this study (Vero, mouse 1D-TK⁺) were able to grow at the same rate when cultured in the presence of 2 mM s⁴T or 2 mM thymidine, albeit at a slower rate than control cells. Consistent with this finding, viruses grown in the presence of1–4 mM s⁴T were obtained in reduced yield but retained full infectivity. Both viruses were specifically inactivated by irradiation with 365 nm light and their photosensitivity, as measured by the initial slope of the inactivation curve, increased in parallel with the concentration of s⁴T added to the culture medium. More than 90% of vaccinia virus grown in the presence of 4 mM s⁴T was inactivated. Organomercurial agarose chromatography of sheared DNA isolated from vaccinia virus grown in the presence of 2 mM s⁴T showed that approximately 2.5% of DNA fragments were specifically retained, as compared to 0.2% for control DNA. This value corresponds to at least one s4T residue incorporated per 30 000 nucleotides of vaccinia virus DNA. In fact, it is likely that this ratio is actually approximately 10 times higher because of the incomplete retention of control thiolated oligodeoxynucleotides. The incorporation of s⁴T into vaccinia virus DNA was required for photoinactivation as (1) the expression of a viral or cellular thymidine kinase was required to confer photosensitivity, and (2) virus plaque reduction assays revealed that maximal photosensitivity coincided with the first rounds of viral DNA replication. The photo-inactivated virus was unable to induce detectable synthesis of several early proteins after infection of cells. These data show that s⁴T is incorporated into the DNA of vaccinia virus grown in the presence of the analogue and then behaves as a built-in UVA light photosensitizer.     

THE PHOTOACTIVE ANTIMICROBIAL PROPERTIES OF EUDISTOMINS FROM THE CARIBBEAN TUNICATE Eudistoma olivaceum

Abstract— Five eudistomins, β-carboline derivatives isolated from a Caribbean tunicate, were tested for phototoxicity against several viruses, bacteria, yeast, and mammalian cells. The five compounds showed varying degrees of UVA dependant phototoxicity (i.e. long wavelength UV dependant) against murine cytomegalovirus (MCMV), Sindbis virus (SV) and mouse 3T3 cells, although the relative order of potency was the same for these three organisms. Eudistomin N was the most active (approximately the same as the β-carboline, harmine), while eudistomins M and O were moderately phototoxic, and H and I had little activity. To some degree the relative phototoxicity was correlated with fewer side chain substituents. A similar relative order of phototoxic potency was seen against phage T4, but in this case the magnitude of the effect was considerably reduced, in contrast to harmine. The antibacterial and antifungal activities were not correlated with antiviral effects, and some UVA-independent activities were seen. Thus the eudistomins may possess different mechanisms of action against different organisms, depending upon the presence or absence of UVA.     

ALPHA POLYMERASE INVOLVEMENT IN EXCISION REPAIR OF DAMAGE INDUCED BY SOLAR RADIATION AT DEFINED WAVELENGTHS IN HUMAN FIBROBLASTS

Abstract Cultured fibroblasts derived from normal human skin have been irradiated at a series of monochromatic wavelengths throughout the ultraviolet region and exposed to the specific α polymerase inhibitor, aphidicolin (1 μg/m l , 2 days) prior to assay for colony forming ability. Repair of 75-80% of the lethal damage induced by UVC (254 nm) or UVB (302 nm, 313 nm) radiation is inhibited by aphidicolin suggesting that such damage is repaired by a common α polymerase dependent pathway. Exposure to aphidicolin after irradiation at longer UVA (334 nm, 365 nm) or a visible (405 nm) wavelength leads to slight protection from inactivation implying that the processing of damage induced in this wavelength region is quite distinct from that occurring at the shorter wavelengths and does not involve α polymerase.     

THE ANTI-HIV ACTIVITIES OF PHOTOACTIVE TERTHIOPHENES

Abstract— Various synthetic analogues of the naturally occurring terthiophene, α-terthienyl (αT), were evaluated for anti-human immunodeficiency virus (HIV) activity. The compounds were incubated individually with a known amount of the virus, with or without UVA radiation (long-wavelength ultraviolet) and residual virus was monitored for its ability to produce cytopathic effects in cell culture and the production of virus-specific protein (p24). The basic terthiophene structure was essential for good anti-HIV activity, although various side chains, such as alcohols, bromo, methyl, thiomethyl and trimethylsilyl groups, permitted retention of maximum activity. Under optimum conditions, as little as 12 ng/mL of these compounds ( i.e approximately 3 × 10⁻⁸ M ) could inactivate 10³ infectious virions. None of the compounds however were more active than αT itself. In all cases, UVA radiation was essential. Several side chains decreased the antiviral efficacy, and some side chains abrogated the activity.     

Photochemical inactivation of alpha- and poxviruses

The objective of this study was to determine whether photochemical inactivation of viruses could be accomplished with high efficiency while preserving the molecular integrity of viral targets allowing subsequent diagnostic tests to be performed at a lower level of containment and cost. We studied the effect of 5-iodonaphthyl 1-azide (INA) and amotosalen (AMO, also known as S-59), which are photochemicals known to target either viral proteins or nucleic acids, respectively. We found that vaccinia virus (VACV, an orthopox virus with a DNA genome) and pixuna virus (PIXV, an alphavirus with an RNA genome) were stable when irradiated with UVA alone or when exposed to either INA or AMO in the dark. AMO followed by UVA exposure was at least 1000-fold more virucidal than INA/UVA on vaccinia and pixuna viruses treated under similar conditions. Photoinactivation with either INA or AMO at conditions that abolished viral infectivity resulted in only minimal impairment of subsequent ELISA and PCR testing. The results presented in this study should assist in developing methods to inactivate in the field environmental and forensic samples suspected of viral contamination, thus limiting the need for costly security and safety operations after an accidental or intentional viral release.     

FURTHER STUDIES ON THE ANTIVIRAL ACTIVITY OF HARMINE, A PHOTOACTIVE β-CARBOLINE ALKALOID

The UV-A mediated antiviral effect of harmine was investigated using murine cytomegalovirus (MCMV) as the target. Virus, which had been inactivated by harmine + UVA, was used to infect cultured mouse cells, and various stages in the viral replication cycle were examined. No viral protein synthesis or RNA synthesis (as measured by polyacrylamide gel electrophoresis or DNA-RNA hybridization) could be detected, and the viral DNA did not replicate (measured by DNA-DNA hybridization). In contrast virus which had been treated with harmine in the dark promoted a complete growth cycle in mouse cells.
An attempt was made to identify the primary target of harmine + UVA activity by examining the bacteriophages T4 and M13, which unlike MCMV do not possess membranes. Both bacteriophages were sensitive, but the single-stranded DNA phage M13 was considerably more so. These results, together with others discussed in the text, suggest that DNA and possibly other macromolecules can serve as targets for harmine photoactivity.     

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.     

Antiviral properties of photosensitizers

Abstract— We have studied the antiviral properties of three different groups of photo-sensitizers, viz. (i) various furyl compounds; (ii) β-carboline alkaloids; (iii) thiophenes and their acetylene derivatives. In general the antiviral potency of the furyl compounds correlated with their ability to produce DNA photoadducts. Among the naturally occurring β-carboline alkaloids, harmine was considerably more potent (in the presence of long wavelength UV radiation, UVA) than several other harmane-related compounds. Slight alterations in chemical structure had profound effects on their antiviral activities. Harmine was shown to inactivate the DNA-virus murine cytomegalovirus (MCMV) by inhibiting viral gene expression, although other targets may also exist. Several eudistomins, carboline derivatives isolated from a tunicate, were also photoactive against viruses. Various plant thiophenes and polyacetylenes were studied in detail. These compounds also required UVA for antiviral activity, and some of them were extremely potent against viruses with membranes, e.g. α-terthienyl, which showed significant activity at only 10^-5μg/ml. When MCMV had been treated with α-terthienyl plus UVA, the virus retained its integrity and penetrated cells normally; but the virus did not replicate. More than 30 additional thiophenes have recently been evaluated, including many synthetic ones, and some of these are even more potent than a-terthienyl. We believe that certain thiophenes possess potential therapeutic value and should be tested against model virus infections in animals.     

8-METHOXYPSORALEN PLUS UVA INDUCES THE 72 kDa HEAT SHOCK PROTEIN IN ORGAN-CULTURED NORMAL HUMAN SKIN

Abstract The proteins induced by heat and other stressors, called heat shock proteins (HSP) or stress proteins, are considered to play a general role in protection from cellular injury. Exposure to UVA (320400 nm) following application of 8-methoxypsoralen (8-MOP), termed PUVA is commonly used in the field of dermatology. In order to understand the induction of HSP in PUVA-treated human skin, indirect immunofluorescence using a monoclonal antibody specific for the 72 kDa HSP (HSP 72) was carried out in organ-cultured normal human skin that was treated with PUVA. When the organ-cultured skin was treated at 37°C for 1 h with 8-MOP at a final concentration of 10 or 100 μg/mL and exposed to UVA (51.3 kJ/m²), nuclear immunofluorcscence of HSP 72 was detected in the epidermal cells 12 h after UVA irradiation. In contrast, the induction of HSP 72 was not detected either by UVA irradiation or 8-MOP treatment. These results suggest that PUVA treatment is one of the stressors for human skin, and DNA damage caused by PUVA induces HSP 72.     

DIFFERENTIAL EFFECTS OF PHOTOACTIVE FURANYL COMPOUNDS ON VIRUS FUNCTIONS

Abstract— Five photoactive furanyl compounds were investigated for their activities against viruses. The two furanocoumarins used were 8-methoxypsoralen (8-MOP) and angelicin; two furanochromones, visnagin and khellin, and the furanoquinoline, dictamnine, were also used. The DNA-containing herpes virus murine cytomegalovirus (MCMV) and the RNA-containing togavirus, Sindbis virus, were the target viruses. All five compounds inactivated both viruses in the presence of UVA, although Sindbis virus was much less sensitive. The relative order of antiviral potency was 8-MOP > dictamnine > visnagin > angelicin > khellin. Dictamnine however was slightly more effective than 8-MOP against Sindbis virus. None of the treatments affected the structural integrity of MCMV, nor did they interfere with the normal transit of the virus into host cells or the localisation of the viral genome in the cell nucleus. Some early viral gene functions were expressed but the viruses did not replicate.     

MODE OF ACTION OF α-TERTHIENYL ON ESCHERICHIA COLI: EVIDENCE FOR A PHOTODYNAMIC EFFECT ON MEMBRANES

The photodynamic effects of α-terthienyl (αT) in near-UV light (UV-A) on Escherichia coli showed close agreement with the light absorption of αT at different wavelengths suggesting that αT is the primary absorbing molecule responsible for the photosensitized reaction. Studies with DNA repair deficient mutants of E. coli indicated that the bactericidal action of αT/UV-A was not mediated by DNA damage, in direct contrast to the well-known photosensitizer, 8-methoxypsoralen. By using a closed borosilicate glass reaction vessel and various gas mixtures, it was demonstrated that photosensitization of both E. coli and a more resistant bacterium, Pseudomonas aeruginosa , was absolutely dependent on the presence of oxygen. The rate of killing by αT/UV-A showed a rather small dependence on preincubation temperatures, with quite rapid killing at 5°C, suggesting that the movement of αT across the cytoplasmic membrane of E. coli is not the rate limiting step in killing and perhaps is not even necessary for killing. Sodium dodecyl sulphate-polyacrylamide gels of cell membrane proteins after 15 and 30min of treatment with αT/UV-A showed substantial random crosslinking of these proteins. The results taken overall suggest that αT is a photodynamic photosensitizer which exerts its primary effect at the level of the cytoplasmic membrane.     

Inactivation of Trypanosoma cruzi Trypomastigote Forms in Blood Components with a Psoralen and Ultraviolet A Light

Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4'-aminomethyl-4,5',8-trimethylpsor-alen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 μg/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type II photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi , the trypomastigote, was achieved at a UVA (320–400 nm radiation) fluence of 4.2 J/cm². We note that while the infectivity of the parasite is eliminated at 4.2 J/cm^Z the trypomastigote motility continues for at least 16 h post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of ³H-AMT indicated that at the lethal UVA fluence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology, which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi , the causative agent of Chagas disease.     

KINETICS OF SIMIAN VIRUS 40 AND LAMBDA INACTIVATION BY PHOTOADDITION OF PSORALEN DERIVATIVES

Abstract The kinetics of psora/en photoinactivation of two distinct DNA viruses, bacteriophage λ and the papovavirus SV40 were investigated. When λ is treated with near ultraviolet light (UVA, 320-400 nm) and 4,5',8-trimethylpsoralen (TMP) at 1 μg/m/, the phage is rapidly inactivated. The survival curve exhibits a distinct shoulder indicating second or higher-order kinetics. SV40, on the other hand, is much more resistant to psoralen photoinactivation and the survival curve is linear, reflecting first order or'pseudo-first order'kinetics. Two TMP derivatives with increased solubility in aqueous solutions, 4'-aminomethyl-TMP and 4'-hydroxymethyl-TMP, were similarly tested. In both virus systems, TMP was much more effective. In experiments designed to examine the role of psoralen cross-link formation in virus inactivation, treated samples were irradiated a second time in the absence of drug. Since reirradiation causes a decline in λ infectivity as great as that observed in continuously irradiated samples, cross-links are implicated as the primary lethal event. In the case of SV40, the results of such a protocol suggest that both monoadducts and cross-links may be lethal or that monoadduct formation may be rate-limiting.     

Mechanical characterization of HIV‐1 with a solid‐state nanopore sensor

Enveloped viruses fuse with cells to transfer their genetic materials and infect the host cell. Fusion requires deformation of both viral and cellular membranes. Since the rigidity of viral membrane is a key factor in their infectivity, studying the rigidity of viral particles is of great significance in understating viral infection. In this paper, a nanopore is used as a single molecule sensor to characterize the deformation of pseudo‐type human immunodeficiency virus type 1 at sub‐micron scale. Non‐infective immature viruses were found to be more rigid than infective mature viruses. In addition, the effects of cholesterol and membrane proteins on the mechanical properties of mature viruses were investigated by chemically modifying the membranes. Furthermore, the deformability of single virus particles was analyzed through a recapturing technique, where the same virus was analyzed twice. The findings demonstrate the ability of nanopore resistive pulse sensing to characterize the deformation of a single virus as opposed to average ensemble measurements.     

Synthesis and specific influenza A virus‐adsorptive functionality of alkyl curdlan sulfate‐coated membrane filter

To develop a biomaterial with an influenza virus‐adsorptive functionality, an alkyl curdlan sulfate was prepared by ionic interaction between a positively charged didodecyldimethyl ammonium bromide and a negatively charged sulfate group of curdlan sulfate, which has potent anti‐HIV activity, and then it was coated on a membrane filter with a 1‐μm pore size by hydrophobic interaction with the long alkyl groups in the curdlan sulfate. The alkyl curdlan sulfate with the degree of alkylation (DOA) of 0.03 (one didodecyldimethyl group/12 sugar residues with 36 hydroxyl or sulfate groups) showed potent anti‐HIV activity in a 50% effective concentration (EC₅₀) as low as 0.87 μg/mL (standard curdlan sulfate EC₅₀ = 0.3 μg/mL), and the activity decreased with increasing DOA. A DOA higher than 0.1 (one didodecyldimethyl group/three sugar residues with nine hydroxyl or sulfate groups) gave no anti‐HIV activity. Although both curdlan sulfate and alkyl curdlan sulfates did not inhibited infection of Madin‐Darby canine kidney cells by influenza viruses, the alkyl curdlan sulfate‐coated membrane filter was found to have a specific adsorptive functionality for influenza A virus in vitro. When 1.6 mg of the alkyl curdlan sulfate with the DOA of 0.03 was coated on a membrane filter (φ13 mm; pore size, 1 μm), three stacked alkyl curdlan sulfate‐coated membrane filters dramatically decreased hemagglutination to 1/4–1/32. However, the membrane filter did not effectively remove on influenza B viruses, and thus a membrane filter without alkyl curdlan sulfate was not effective against influenza viruses. These results can therefore be presumed to demonstrate that the alkyl curdlan sulfate‐coated membrane filter removed influenza A viruses by adsorption between the negatively charged sulfate groups and the positively charged envelope protein of the virus. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

ANTIVIRAL EFFECT OF HARMINE, A PHOTOACTIVE β-CARBOLINE ALKALOID

Abstract— Harmine, a β-carboline alkaloid, was found to be an efficient photoactive agent against the DNA-containing murine cytomegalovirus (MCMV) and the RNA containing Sindbis virus, a togavirus, both of which contain membranes. In contrast the host cells themselves were relatively resistant. The MCMV was investigated in more detail. Virus which had been inactivated by harmine plus UVA retained its structural integrity and was fully capable of penetrating host cells, following which the viral genome entered the cell nucleus. The viral genes which normally code for the switch-off in thymidine kinase activity arid for the normal cytopathic effects were not expressed; however the viral geneis responsible for the cytotoxic effect due to high multiplicities of infection was still expressed, indicating that there may be a differential block of some viral genes     

NON-NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m² for the UVASUN lamp, 75 to 390 J/m² for the FS20 lamp and 3.8 to 17.2 J/m² for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.     

Virus rejection with two model human enteric viruses in membrane bioreactor system

A membrane bioreactor (MBR) with gravity drain was tested for virus rejection with two coliphages, T4 and f2, which were used as surrogates for human enteric viruses. Virus rejection was investigated by PVDF and PP membrane modules, with the pore sizes of 0.22 and 0.1 μm, respectively. In tap water system, 2.1 lg rejection of coliphage T4 could be achieved by PVDF membrane compared with complete rejection by PP membrane, while for coliphage f2 with smaller diameter, 0.3―0.5 lg rejection of the influent virus was removed by the two membranes. In domestic wastewater system, cake layer and gel layer on the membrane surface changed the cut-off size of the membrane so that there was no significant difference between PP and PVDF for each coliphage. The removal ratios of coliphage T4 and f2 in the MBR were more than 5.5 and 3.0 lg, respectively. Compared with 5.5 lg removal for virus T4 in the MBR system, only 2.1 lg (96.8%―99.9%) removal rate was observed in the conventional activated sludge system with the influent virus concentration fluctuating from 1830 to 57000 PFU/mL. Only 0.8%―22% virus removal was the effect of adsorption to activated sludge, which showed a decreasing tendency with the retention time, while 75%―98% was the effect of virus inactivation by microbial activity. It indicated that the major mechanism of virus removal was not the transfer of viruses from the water phase to the sludge phase but inactivation in the biological treatment process.