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.     

Duck Hepatitis B Virus Inactivation and 8-Methoxypsoralen Photoadduct Formation in Human Platelet Concentrates*

Photochemical inactivation (PCI) of virus and bacteria in platelet concentrates (PC) has been demonstrated using 8-methoxypsoralen (8-MOP) and long-wavelength UV light (UVA). To study inactivation of blood-borne virus, we have employed duck hepatitis B virus (DHBV), a model for human hepatitis B virus. A specific hepatocyte culture infectivity assay, with PCR detection, could measure 5–6 log₁₀ virus kill. The DHBV inactivation in PC was dependent on UVA dose, was enhanced when plasma was reduced from 100% to 20% and was limited by 8-MOP solubility in the reduced-plasma medium. Optimum conditions for PCI were 100 μg/mL 8-MOP in 20% plasma and 80% synthetic platelet storage medium. A radiolabeling assay for 8-MOP photoadducts in hepatocytes seeded into PC confirmed that DHBV inactivation reflected DNA modification and indicated that adduct formation was insensitive to minor variations in conditions. Kinetic modeling indicated that optimum adduct formation was a compromise between 8-MOP dark binding and optical transmittance and that plasma proteins competed for 8-MOP binding. The PCI results in various media correlated with corresponding DNA modification densities and were compared to statistical models incorporating DHBV characteristics and predictions of 8-MOP crosslink formation between DNA strands. Behavior was consistent with one or a small number of lethal modifications per DNA strand, including monoadducts, but probably not crosslinks alone. A minor subpopulation of DHBV was found to be, somewhat more difficult to inactivate, consistent with three-fold lower modification, due possibly to single-stranded DNA character or host repair of photoadducts.     

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.     

3′-(1,2,3-Triazol-1-yl)-2′,3′-dideoxythymidine and 3′-(1,2,3-triazol-1-yl)-2′,3′-dideoxyuridine

Cycloaddition of different acetylenic compounds on the azido function of 3′-azido-2′,3′-dideoxythymidine and 3′-azido-2′,3′-dideoxyuridine afforded products with a 1,2,3-triazol-1-yl substituent in the 3′-position. In contrast with the parent compounds, these triazolyl derivatives had no appreciable activity against human immunodeficiency virus (HIV-1).     

Synergistic Effect by Combining a gp120-Binding Protein and a gp41-Binding Antibody to Inactivate HIV-1 Virions and Inhibit HIV-1 Infection

Acquired immune deficiency syndrome (AIDS) has prevailed over the last 30 years. Although highly active antiretroviral therapy (HAART) has decreased mortality and efficiently controlled the progression of disease, no vaccine or curative drugs have been approved until now. A viral inactivator is expected to inactivate cell-free virions in the absence of target cells. Previously, we identified a gp120-binding protein, mD1.22, which can inactivate laboratory-adapted HIV-1. In this study, we have found that the gp41 N-terminal heptad repeat (NHR)-binding antibody D5 single-chain variable fragment (scFv) alone cannot inactivate HIV-1 at the high concentration tested. However, D5 scFv in the combination could enhance inactivation activity of mD1.22 against divergent HIV-1 strains, including HIV-1 laboratory-adapted strains, primary HIV-1 isolates, T20- and AZT-resistant strains, and LRA-reactivated virions. Combining mD1.22 and D5 scFv exhibited synergistic effect on inhibition of infection by divergent HIV-1 strains. These results suggest good potential to develop the strategy of combining a gp120-binding protein and a gp41-binding antibody for the treatment of HIV-1 infection.     

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.     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

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.     

Temperature inducible beta-sheet structure in the transactivation domains of retroviral regulatory proteins of the Rev family

The interaction of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev with cellular cofactors is crucial for the viral life cycle. The HIV-1 Rev transactivation domain is functionally interchangeable with analog regions of Rev proteins of other retroviruses suggesting common folding patterns. In order to obtain experimental evidence for similar structural features mediating protein-protein contacts we investigated activation domain peptides from HIV-1, HIV-2, VISNA virus, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) by CD spectroscopy, secondary structure prediction and sequence analysis. Although different in polarity and hydrophobicity, all peptides showed a similar behavior with respect to solution conformation, concentration dependence and variations in ionic strength and pH. Temperature studies revealed an unusual induction of beta-structure with rising temperatures in all activation domain peptides. The high stability of beta-structure in this region was demonstrated in three different peptides of the activation domain of HIV-1 Rev in solutions containing 40% hexafluoropropanol, a reagent usually known to induce alpha-helix into amino acid sequences. Sequence alignments revealed similarities between the polar effector domains from FIV and EIAV and the leucine rich (hydrophobic) effector domains found in HIV-1, HIV-2 and VISNA. Studies on activation domain peptides of two dominant negative HIV-1 Rev mutants, M10 and M32, pointed towards different reasons for the biological behavior. Whereas the peptide containing the M10 mutation (L78E79-->D78L79) showed wild-type structure, the M32 mutant peptide (L78L81L83-->A78A81A83) revealed a different protein fold to be the reason for the disturbed binding to cellular cofactors. From our data, we conclude, that the activation domain of Rev proteins from different viral origins adopt a similar fold and that a beta-structural element is involved in binding to a cellular cofactor.     

Comparison of the efficiency and the specificity of DNA-bound and free cationic porphyrin in photodynamic virus inactivation

The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to remove non-enveloped viruses from aqueous media. Here we tested the virus inactivation capability of meso-Tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4-N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be efficient photosensitizers of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding.     

UVA- and UVB-induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice.

The modifications induced in hairless mouse skin by chronic UV irradiation were investigated. Skin explant cultures were used to study UVA- and UVB-induced changes occurring in interstitial collagen (type I and type III) and fibronectin biosynthesis. To study the long-term effects, albino hairless mice were irradiated with UVA radiation alone from two sources with different spectral qualities or with UVB. UVA and UVB radiation produced a significant increase in the ratio of type III to type I collagen (more than 100% for UVA-irradiated skin and about 60% for UVB-irradiated skin) accompanied by a significantly increased fibronectin biosynthesis (50% or more in all irradiated groups). Irradiation with either UVA or UVB alone had no significant effect on the total collagen synthesis and resulted in only a slight decrease in the total collagen content of the skin determined as hydroxyproline. This decrease was significant only in the case of the group irradiated with UVA (xenon) (decrease of 25%, expressed as micrograms of hydroxyproline per milligram wet weight). A significant decrease in collagen hydroxylation (expressed as radioactive hydroxyproline/radioactive hydroxyproline plus proline in neosynthesized collagen) was observed of about 50% in skin irradiated with UVA (xenon) but not in UVB-treated skin. Several of the above modifications (increased fibronectin biosynthesis, increased collagen type III to type I ratio) correspond to the modifications observed during the aging of non-irradiated hairless mice. Therefore it appears that UV irradiation accelerates the modifications of extracellular matrix biosynthesis observed during aging.     

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.     

Photochemical inactivation of Pseudomonas aeruginosa

Adaptability to a broad range of environments together with relatively high resistance to antibiotics and to disinfectants makes Pseudomonas aeruginosa a concern in hospitals and in public health. We investigated whether UVA-mediated photochemical inactivation of P. aeruginosa could be accomplished with high efficiency while at the same time preserving the sensitivity of subsequent diagnostic tests. We characterized dose responses and bactericidal kinetic rates of 5-iodonaphthyl 1-azide (INA) and of amotosalen (AMO) as these substances exposed to UVA are known to inactivate germs with minimal impact to blood products or to viral antigens. Neither UVA without photochemicals nor INA or AMO in the dark inactivated bacteria. We found that AMO was ca 1000-fold more effective in inactivating P. aeruginosa cells than INA under similar conditions. Photoinactivation with either INA or AMO at conditions that abolished bacterial infectivity did not impair polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) testing. For comparison, similar titers of Bacillus atrophaeus spores (a surrogate for B. anthracis) remained unaffected at conditions that reduced the survival of P. aeruginosa below detection levels. The results presented in this study should assist in improved methods to inactivate P. aeruginosa in environmental, clinical and forensic samples without impairing subsequent nucleic acid- or immune-based analysis.     

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.     

ANTIVIRAL ACTIVITY OF THE PHOTOACTIVE THIOPHENE α-TERTHIENYL

The naturally occurring thiophene, α-terthienyl, was investigated for phototoxicity against several viruses and a line of mouse cells. The compound was extremely phototoxic to the two-membrane-containing animal viruses, murine cytomegalovirus (MCMV) and Sindbis virus (SV). Antiviral activity was detected at 10⁵μg/m in the presence of UVA. However, no effect was seen in the absence of UV-A, even at 0.1 μg/m of αT. Mouse cells were much more resistant to αT, as was the bacterial virus T4, which does not contain a membrane. Murine CMV, which had been inactivated by αT and UVA, penetrated mouse cells efficiently; but the viral DNA could not replicate, and late viral proteins were not made. Thus viral gene expression was inhibited in the photoinactivated virus. In order to account for all these data we suggest that αT may interact with viral proteins in addition to membrane lipids.     

Induction of apoptosis by photoexcited tetracyclic compounds derivatives of benzo[b]thiophenes and pyridines

The antiproliferative activity, upon UVA irradiation, of two tetracyclic derivatives of benzo[b]thiophenes and pyridines, a benzo[b]thienopyridopyrimidone (1) and a thienocarboline (2), has been investigated in a panel of human tumor cell lines. The two compounds present a remarkable cytotoxicity after UVA irradiation (365 nm), reaching an IC50 of 0.1 microM in the leukaemia cell lines and 0.3-0.5 microM in the solid tumour cell lines. Their effect on the cell cycle was measured by flow cytometry in Jurkat cells. The compounds induce cell cycle perturbations and trigger a massive apoptosis as revealed by the externalisation of Annexin V-targeted residues at the outer plasmatic membrane. Furthermore the drugs induce, upon UVA irradiation significant variations of the mitochondrial potential (Deltapsi(mt)) measured by flow cytometry using the fluorochrome JC-1. In addition we characterized the mitochondrial production of reactive oxygen species (ROS) using the probe dihydroethidine (HE) and the oxidations of the mitochondrial phospholipid cardiolipin using the interacting probe nonyl acridine orange (NAO). Both compounds stimulate the production of ROS, and remarkably induce oxidation of cardiolipin. We have investigated the DNA-binding properties of these two compounds by means of UV-Vis spectroscopy and fluorescence. The two compounds exhibit a low affinity toward the macromolecule. The mode of binding was also investigated by means of flow linear dichroism (LD) which has revealed that the two compounds do not efficiently intercalate into DNA. Finally, the DNA-photocleavaging properties of the test compounds were studied on pBR322 plasmid DNA as a model. Only compound 1 is able to induce a significant production of single strand breaks only after digestion with the base excision repair enzyme Endo III. Altogether these data suggest that DNA is not a preferential target of these molecules and other subcellular structures may be responsible for their high phototoxic activity.     

Phototoxicity against tumor cells and Sindbis virus by an octahedral rhodium bisbipyridyl complex and evidence for the genome as a target in viral photoinactivation

An octahedral rhodium complex (cis-dichloro(dipyrido[3,2a-2',3'c]phenazine)(1,10-phenanthroline)rhodium(III) chloride; DPPZPHEN) has been prepared that can penetrate tumor cell membranes and the Sindbis viral capsid. The compound is phototoxic to these entities when irradiated with UVA light. Model studies with calf thymus and supercoiled plasmid DNA indicate that the complex can both bind with, and nick, nucleic acid. Analysis of Sindbis virus, following irradiation with the metal complex, confirmed that the viral genome was rendered noninfectious by this treatment.     

Disinfection and Mechanistic Insights of Escherichia coli in Water by Bismuth Oxyhalide Photocatalysis

This study demonstrates the potential of a new BiOCl_0.875Br_0.125 photocatalyst to disinfect Escherichia coli in water under simulated solar irradiation. Photocatalytic efficiency was examined for different photocatalyst loadings, solar wavelengths, exposure times, photocatalyst concentration × contact time (Ct) concept and with the use of scavengers. To elucidate the inactivation mechanism, we examined DNA damage, membrane damage, lipid peroxidation and protein release. Both photolysis and photocatalysis were negligible under visible irradiation, but enhanced photocatalytic activity was observed under solar UVA (λ > 320 nm) and UVB (λ > 280 nm), with 1.5 and 3.6 log inactivation, respectively, after 40 min of irradiation. The log inactivation vs Ct curve for E. coli by UVA/BiOCl_0.875Br_0.125 was fairly linear, with Ct = 10 g L^?1 × min, resulting in 2 log inactivation. Photocatalytic treatment led to membrane damage, but without lipid peroxidation. Accordingly, protein was released from the cells after UVA or UVA/BiOCl_0.875Br_0.125 treatment. Photocatalysis also increased endonuclease‐sensitive sites vs photolysis alone, by an unknown mechanism. Finally, E. coli inactivation was not influenced by the addition of tert‐butanol or l ‐histidine, implying that neither hydroxyl radicals nor singlet oxygen reactive species are involved in the inactivation process.