Photodynamic Inactivation of Human Coronaviruses.

Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species (ROS) that can inactivate microorganisms. The botanical extract PhytoQuinTM is a powerful photosensitizer with antimicrobial properties. We previously demonstrated that photoactivated PhytoQuin also has antiviral properties against herpes simplex viruses and adenoviruses in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. Here, we report that human coronaviruses (HCoVs) are also susceptible to photodynamic inactivation. Photoactivated-PhytoQuin inhibited the replication of the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 in cultured cells across a range of sub-cytotoxic doses. This antiviral effect was light-dependent, as we observed minimal antiviral effect of PhytoQuin in the absence of photoactivation. Using RNase protection assays, we observed that PDI disrupted HCoV particle integrity allowing for the digestion of viral RNA by exogenous ribonucleases. Using lentiviruses pseudotyped with the SARS-CoV-2 Spike (S) protein, we once again observed a strong, light-dependent antiviral effect of PhytoQuin, which prevented S-mediated entry into human cells. We also observed that PhytoQuin PDI altered S protein electrophoretic mobility. The PhytoQuin constituent emodin displayed equivalent light-dependent antiviral activity to PhytoQuin in matched-dose experiments, indicating that it plays a central role in PhytoQuin PDI against CoVs. Together, these findings demonstrate that HCoV lipid envelopes and proteins are damaged by PhytoQuin PDI and expands the list of susceptible viruses.

Application of a Photocatalyst as an Inactivator of Bovine Coronavirus.

Bovine coronavirus (BCoV), a major causative pathogen of bovine enteric and respiratory diseases and a zoonotic pathogen transmissible between animals and humans, has led to severe economic losses in numerous countries. BCoV belongs to the genus Betacoronavirus, which is a model of a pathogen that is threatening human health and includes severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus. This study aimed to determine whether photocatalytic material effectively reduces CoVs in the environment. Using the film adhesion method of photocatalytic materials, we assessed its antiviral activity and the effect of visible light irradiation according to methods defined by the International Organization for Standardization. Consequently, photocatalytic material was found to have antiviral activity, reducing the viral loads by 2.7 log TCID50 (tissue culture infective dose 50)/0.1 mL (500 lux), 2.8 log TCID50/0.1 mL (1000 lux), and 2.4 log TCID50/0.1 mL (3000 lux). Hence, this photocatalytic material might be applicable not only to reducing CoVs in the cattle breeding environment but also perhaps in other indoor spaces, such as offices and hospital rooms. To our knowledge, this study is the first to evaluate the antiviral activity of a photocatalytic material against CoV.

Laser irradiated phenothiazines: New potential treatment for COVID-19 explored by molecular docking.

The worldwide infection with the new Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) demands urgently new potent treatment(s). In this study we predict, using molecular docking, the binding affinity of 15 phenothiazines (antihistaminic and antipsychotic drugs) when interacting with the main protease (Mpro) of SARS-CoV-2. Additionally, we tested the binding affinity of photoproducts identified after irradiation of phenothiazines with Nd:YAG laser beam at 266 nm respectively 355 nm. Our results reveal that thioridazine and its identified photoproducts (mesoridazine and sulforidazine) have high biological activity on the virus Mpro. This shows that thioridazine and its two photoproducts might represent new potent medicines to be used for treatment in this outbreak. Such results recommend these medicines for further tests on cell cultures infected with SARS-CoV-2 or animal model. The transition to human subjects of the suggested treatment will be smooth due to the fact that the drugs are already available on the market.

Photolysis of three antiviral drugs acyclovir, zidovudine and lamivudine in surface freshwater and seawater.

Photodegradation is an important elimination process for many pharmaceuticals in surface waters. In this study, photodegradation of three antiviral drugs, acyclovir, zidovudine, and lamivudine, was investigated in pure water, freshwater, and seawater under the irradiation of simulated sunlight. Results showed that zidovudine was easily transformed via direct photolysis, while acyclovir and lamivudine were mainly transformed via indirect photolysis. We found that in freshwater, nitrate enhanced the photodegradation of the three antiviral drugs, bicarbonate promoted the photodegradation of acyclovir, and dissolved organic matter (DOM) accelerated the photolysis of acyclovir and lamivudine. In seawater, the photolysis of acyclovir was not susceptible to Cl(-), Br(-) and ionic strength; however, the photolysis of zidovudine was inhibited by Cl(-) and Br(-), and the photolysis of lamivudine was enhanced by Cl(-), Br(-) and ionic strength. Second-order reaction rate constants for the three antiviral drugs with (1)O2 (k1O2) and OH (kOH) were also measured. These results are important for fate and ecological risk assessment of the antiviral drugs in natural waters.

Photocatalytic degradation of the antiviral drug Tamiflu by UV-A/TiO2: Kinetics and mechanisms.

The photocatalytic degradation of the antiviral drug Tamiflu (oseltamivir phosphate, OP) by TiO2 - P25, ST-01 and ATO was investigated in aqueous solution under ultraviolet (UV-A) irradiation. The photocatalysis of OP is well described by pseudo-first-order kinetics with r2>98.0% for all cases. The kinetic constant of P25 with 80% anatase and 20% rutile (0.040 min(-1)) is 4 and 10 times higher than that of ATO and ST-01 with 100% purity of anatase, respectively. We examined the effects of the catalyst loading and initial OP concentration on the photodegradation of OP, and used potassium iodine, isopropanol, and calcium fluorine as radical quenchers to evaluate the contributions of the hydroxyl radical (OH) and photo hole (h+) in the photodegradation. Results confirmed that 80% of the contribution came from the OH species. Although more than 95% of the OP (21 µM) was removed after 80 min of UV-A irradiation with 20 and 100 mg L(-1) P25, the removal efficiencies of total organic carbon (TOC) were only 45.6% and 67.0%, respectively, after 360 min UV-A irradiation. Based on an intermediate analysis by HPLC coupled with a triple quadrupole spectrometer and an ion trap mass spectrometer, typical intermediate species such as hydration derivatives, hydroxyl substitutes and keto-derivatives were identified and possible degradation pathways of OP by P25 were proposed.

Evaluation of the phototransformation of the antiviral zanamivir in surface waters through identification of transformation products.

The antiviral zanamivir has been recently reported to occur in surface waters where its presence may lead to the selection of resistant strains of virus in aquatic fauna. In order to evaluate the fate of zanamivir in surface waters, its susceptibility to phototransformation was evaluated using simulated and natural sunlight. Upon exposure of aqueous solutions (20µgL(-1)) to simulated sunlight, zanamivir in surface water degraded at t1/23.6h. Under natural sunlight in surface water about 30% of the initial concentration of the antiviral disappeared within 18 days. The experiments with surface water showed similar effect as humic acid addition with expected decreasing effect on degradation while nitrate addition showed increasing effect. In the experiments with artificial sunlight at high concentrations of zanamivir, four photoproducts were tentatively identified by hydrophilic interaction chromatography-LTQ-Orbitrap-MS, showing [M+H](+) ions at m/z 112 (TP111), m/z 275 (TP274), m/z 323 (TP322), and m/z 333 (TP332). However at 20µgL(-1) only the formation of the recalcitrant TP111 was observed. The proposed structures were rationalized by photolysis mechanisms. Photoproduct TP111 was confirmed with a commercially available standard (isocytosine). In summary, the findings suggest that the photodegradation of zanamivir in surface waters proceeds with slow kinetics.

Photochemical stability of 4'-azido-2'-deoxy-2'-methylcytidine hydrochloride: structural elucidation of major degradation products by LC-MS and NMR analysis.

The photochemical stability of (1'R,2'S,3'S,4'R)-4'-azido-2'-deoxy-2'-methylcytidine hydrochloride, a new anti-HCV agent, was investigated. Aqueous solutions and bulk drug powder of the drug candidate were exposed to UV-visible light, complying with ICH requirements. The nucleoside analog decomposed via loss of nitrogen to yield products derived from a highly reactive azide intermediate. Major photolysis products were identified by LC-MS and NMR analysis, revealing three main photodegradation pathways. The first one led to the formation of a ring-expanded imidate ester. The other degradation pathways involved exocyclic or endocyclic bond cleavage with imine or imino lactone formation. The latter were prone to rapid hydrolysis, eventually resulting in the release of cytosine, 2-methyl malonaldehyde and (E)-cytosyl-2-methylpropenal.

Light-activated nanotube-porphyrin conjugates as effective antiviral agents.

Porphyrins have been used for photodynamic therapy (PDT) against a wide range of targets like bacteria, viruses and tumor cells. In this work, we report porphyrin-conjugated multi-walled carbon nanotubes (NT-P) as potent antiviral agents. Specifically, we used Protoporphyrin IX (PPIX), which we attached to acid-functionalized multi-walled carbon nanotubes (MWNTs). We decided to use carbon nanotubes as scaffolds because of their ease of recovery from a solution through filtration. In the presence of visible light, NT-P was found to significantly reduce the ability of Influenza A virus to infect mammalian cells. NT-P may be used effectively against influenza viruses with little or no chance of them developing resistance to the treatment. Furthermore, NT-P can be easily recovered through filtration which offers a facile strategy to reuse the active porphyrin moiety to its fullest extent. Thus NT-P conjugates represent a new approach for preparing ex vivo reusable antiviral agents.

Novel 3-sulphonamido-quinazolin-4(3H)-one derivatives: microwave-assisted synthesis and evaluation of antiviral activities against respiratory and biodefense viruses.

We designed and synthesized novel 2,3-disubstituted quinazolin-4(3H)-ones by microwave technique and characterized them by spectral analysis. Synthesized compounds were screened for cytotoxicity and for antiviral activity against influenza A (H1N1, H3N2 and H5N1), severe acute respiratory syndrome corona, dengue, yellow fever, Venezuelan equine encephalitis (VEE), Rift Valley fever, and Tacaribe viruses in cell culture. A neutral red uptake assay was used to determine 50% virus-inhibitory concentrations (EC50) of test compounds and their 50% cytotoxicity concentration (CC50) in uninfected Madin-Darby canine kidney, Vero, and Vero 76 cells; selectivity indices (ratio of CC50 to EC50) were derived from the data. The compound 4-(6,8-dibromo-4-oxo-2-phenyl quinazolin-3(4H)-yl)-N-(4,5-dimethyloxazol-2yl) benzenesulphonamide 15 inhibited the replication of avian influenza (H5N1) virus (EC50 = 8.4 microg/ml, CC50 > 100 microg/ml, SI > 11.9) as did 4-(6-bromo-4oxo-2phenylquinazolin-3(4H)-yl) benzene]sulphonamide 5 (EC50 = 3 microg/ml, CC50 = 32 microg/ml, SI = 11). Compound 5 was also moderately active against VEE and Tacaribe viruses. The methodology described in this report is applicable for rapid synthesis of many compounds with potential antiviral properties.

Synthesis of cationic beta-vinyl substituted meso-tetraphenylporphyrins and their in vitro activity against herpes simplex virus type 1.

An easy route to cationic beta-vinyl substituted meso-tetraphenylporphyrin derivatives is described. Two novel compounds were tested in vitro for their antiviral photoactivity against herpes simplex virus type 1. One of these compounds exhibited a significant activity, reaching 99% of virus inactivation after 15 min of photoactivation.

Use of a flow-cell system to investigate virucidal dimethylmethylene blue phototreatment in two RBC additive solutions.

BACKGROUND: Limited photoinactivation kinetics, use of low-volume 30 percent Hct RBCs, and hemolysis have restricted the practicality of the use of dimethylmethylene blue (DMMB) and light for RBC decontamination. A flow-cell system was developed to rapidly treat larger volumes of oxygenated 45 percent Hct RBCs with high-intensity red light. MATERIALS AND METHODS: CPD-whole blood was WBC reduced, RBCs were diluted in additive solutions (either Adsol or Erythrosol), and suspensions were subsequently oxygenated by gas overlay. Intracellular or extracellular VSV and DMMB were sequentially added. VSV-infected RBC suspensions (45% Hct) were passed through 1-mm-thick flow cells and illuminated. Samples were titered for VSV, stored for up to 42 days, and assayed for Hb, supernatant potassium, ATP, and MCV. RESULTS: The use of oxygenated RBCs resulted in rapid and reproducible photoinactivaton of > or = 6.6 log extracellular and approximately 4.0 log intracellular VSV independent of additive solution. Phototreated Adsol RBCs exhibited more than 10 times greater hemolysis and 30 percent greater MCV during storage than identically treated Erythrosol RBCs. Phototreatment caused RBC potassium leakage from RBCs in both additive solutions. ATP levels were better preserved in Erythrosol than Adsol RBCs. CONCLUSION: A rapid, reproducible, and robust method for photoinactivating model virus in RBC suspensions was developed. Despite improved hemolysis and ATP levels in Erythrosol-phototreated RBCs, storage properties were not maintained for 42 days.

Influence of radiation sterilization on the stability of trifluorothymidine.

The influence of radiation sterilization on the stability of trifluorothymidine (TFT) was investigated. TFT was irradiated under ambient atmosphere with a 60Co-source and with an electron accelerator at 25, 50, and 100 kGy, respectively. The radiation-induced effects were determined by chromatographic and spectroscopic methods as well as potentiometrically with a fluoride selective electrode. TFT was moderately stable to ionizing radiation. The degradation induced by electron-beam irradiation was significantly (P=95%) smaller than by gamma-irradiation. The radiolysis products amounted to about 0.25% after electron-beam irradiation at 25 kGy, and to about 0.50% after gamma-irradiation, respectively. The main irradiation product was 5-trifluoromethyluracil (TFMU). In addition five further impurities were detected with HPLC. Identification of degradation products was performed using HPLC-ESI-MS. A degradation path of TFT after radiation sterilization was shown.

Is the excited-state H-atom transfer in hypericin concerted?

The excited-state intramolecular H-atom transfer of hypericin (Hyp) was investigated as a function of pH in monodispersed reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane/water and in complexes with Tb3+ under conditions in which one of the two carbonyl groups of Hyp is incapable of accepting a hydrogen atom. The results of pump-probe transient absorption experiments provide no evidence for a concerted H-atom transfer mechanism.

Influence of methylene blue photoinactivation treatment on coagulation factors from fresh frozen plasma, cryoprecipitates and cryosupernatants.

OBJECTIVE: To study the influence of virus photoinactivation with methylene blue (MB) on the coagulation factors of fresh frozen plasma (FFP) and the corresponding cryoprecipitates and cryosupernatants derived from it. MATERIALS AND METHODS: The photoinactivation procedure of the German Red Cross (Springe) was applied using Biomat (Grifols, Spain). Twenty isogroup pools of three plasma units were made from 60 U of FFP. The pools were split into three bags. One of them was photoinactivated, and pre- and postinactivation samples (MB-plasma) were obtained. The second bag was treated in the same way, followed by the preparation of MB-cryoprecipitate and MB-cryosupernatant. The third bag was not photoinactivated, and was processed in the same way to obtain control cryoprecipitate and cryosupernatant. The prothrombin time and activated partial thromboplastin time were analysed, as well as fibrinogen, factors (F) II, V, VII, VIII, IX, XI and XIII, antithrombin III, von Willebrand (vW) F:RCo, vWF:Ag and the multimeric structure of vWF. RESULTS: In plasma, the proteins most sensitive to photoinactivation were fibrinogen, FV, FVIII, FIX and FXI (24, 32, 28, 23 and 27% loss, respectively). In the MB-cryoprecipitate, the losses were higher for FVIII (23%), moderate for fibrinogen, FXIII and vWF:RCo (18, 14 and 13%, respectively) and minimal (only 3%) for vWF:Ag. In MB-cryosupernatants, the losses were higher for FV (26%) and moderate for fibrinogen (16%), FIX (18%) and FXI (19%), as well as for FII and FXIII (15%). The multimeric structure of vWF was not modified in MB-plasma or in MB-cryoprecipitates. The supernatants (both MB treated as well as controls) showed an absence of multimers of very high and high molecular weight. CONCLUSIONS: The quantitative and qualitative conservation of coagulation factors achieved in MB-plasma-derived products suggest that they are useful for the global replacement of coagulation factors and for deficiencies in FV and FXI. In countries lacking the economic resources to obtain virally inactivated concentrates, MB-cryoprecipitates could be useful in von Willebrand's disease and fibrinogen and FXIII deficiencies. MB-cryosupernatants could be employed in thrombotic thrombocytopenic purpura, in the correction of total or partial deficiencies of prothrombin complex factors and in specific deficiencies of FV and FXI.

The role of oxygen in the antiviral activity of hypericin and hypocrellin.

The light-induced antiviral activity of hypericin and hypocrellin in the presence and absence of oxygen was examined under experimental conditions where the effect of oxygen depletion could be quantified. There was a significant reduction of light-induced antiviral activity of hypericin and hypocrellin under hypoxic conditions. Interestingly, antiviral activity of hypocrellin was not observed at low oxygen levels at which hypericin retained measurable virucidal activity. This suggests that additional pathways, such as the generation of protons from excited states of hypericin, may enhance the biological activity of activated oxygen species.

Antiviral substance from silkworm faeces: purification and its chemical characterization.

In a previous paper, we reported that an extract of silkworm faeces has a marked antiviral activity on enveloped viruses, but not on a non-enveloped virus, and we showed that it inhibits the synthesis of a viral specific gene of HVJ (Sendai virus) without affecting the viral adsorption and entry into the host cell. In this paper, we accomplished the purification of an antiviral active substance by extraction of a hydrophobic substance and thin layer chromatography. The active substance was found to be a chlorophyll-like substance with a molecular mass of about 530. This substance shows clear antiviral activity against HVJ, HSV (herpes simplex virus type-1), and HIV (human immunodeficiency virus type-1). Its antiviral activity was dependent on light irradiation and temperature. Furthermore, it also possesses a strong hemolytic activity under light.

Chemiluminescent activation of the antiviral activity of hypericin: a molecular flashlight.

Hypericin is a naturally occurring photosensitizer that displays potent antiviral activity in the presence of light. The absence of light in many regions of the body may preclude the use of hypericin and other photosensitizers as therapeutic compounds for the treatment of viral infections in vivo. The chemiluminescent oxidation of luciferin by the luciferase from the North American firefly Photinus pyralis was found to generate sufficiently intense and long-lived emission to induce antiviral activity of hypericin. Light-induced virucidal activity of hypericin was demonstrated against equine infectious anemia virus, a lentivirus structurally, genetically, and antigenically related to the human immunodeficiency virus. The implications for exploiting chemiluminescence as a "molecular flashlight" for effecting photodynamic therapy against virus-infected cells and tumor cells are discussed.

Inhibition of retrovirus-induced syncytium formation by photoproducts of a brominated 1,8-naphthalimide compound.

A major disadvantage of conventional phototherapy is the requirement for the in situ delivery of stimulating photoenergy subsequent to the binding of photochemicals to target malignant cells, or virus-infected cells, or viruses. This drawback has resulted in considerable limitation in the use of photochemicals in photomedicine. To circumvent this problem, we have investigated the antiviral efficacy of a brominated 1,8-naphthalimide photocompound, termed LY66Br [3-bromo-4-(hexylamino)-N-hexyl-1,8-naphthalimide], which upon exposure to visible light at 420 nm generates independently of oxygen one or more stable antiviral molecular photoproducts (e.g., is 'preactivated'). Human cell lines infected with the human immunodeficiency virus type 1 (HIV-1), or with the human T-lymphotropic virus type-1 (HTLV-I) exposed to photochemical products of LY66Br (P-LY66Br) completely lost their ability to form syncytia in vitro. Photoproducts of P-LY66Br retain full antiviral activity for at least 3 and 6 weeks when stored at room temperature and at -80 degrees C, respectively. Concentrations of P-LY66Br, effective in inhibiting syncytium formation mediated by HIV-1 and HTLV-I, were nontoxic to normal red cell components of whole blood (red blood cell 2,3-diphosphoglyceric acid, adenosine triphosphate, osmotic fragility or blood type antigens). Additionally, no evidence of acute toxicity was demonstrated in mice following an intravenous bolus inoculation to achieve plasma concentration of 600 microM of P-LY66Br. These findings represent the first demonstration of inhibition of retrovirus-induced syncytium formation by a photochemical product, and justify further investigation of the preactivation process of photochemicals in the treatment of systemic viral infections such as the acquired immunodeficiency syndrome (AIDS), in cancer therapy, and in sterilization of banked blood products.

Photodynamic inactivation of simian immunodeficiency virus.

A photodynamic flow system employing a dihematoporphyrin ether (DHE) was tested for its ability to inactivate the in vitro infectivity of simian immunodeficiency virus (SICMac) at 630 +/- 5 nm with a light fluence of 5 J/cm2. Cell-free SIVMac was inactivated by photoactivated hematoporphyrin derivative in a dose-dependent fashion. Since SIVMac is closely related to human immunodeficiency virus type 2 (HIV-2) and we have previously reported the successful photodynamic inactivation of HIV-1 in cell-free medium as well as in whole human blood, this technology has the potential for the eradication of transfusion-associated acquired immunodeficiency diseases caused by the above-mentioned retroviruses.

Effect of storage and 60Co irradiation on the antiviral activity and subtype pattern of native human interferon-alpha preparations.

The stability of native human leukocyte IFN-alpha was investigated after one year storage at different temperatures, or after sterilization with 60Co-irradiation by determining the total antiviral activity of samples. Inactivation of VSV and Aujeszky virions by 60Co-irradiation was directly related to the radiolysis of samples, indicating the uselessness of this procedure for sterilization of IFN-alpha preparations. The presence and proportion of subtypes in the stored or irradiated preparations (with 50 and 25-70% inactivation, respectively) was analysed by chromatofocusing, comparing their patterns with that of the untreated controls. A logarithmic correlation was found between the pI values and temperature/irradiation sensitivity of subtypes.