PHOTODYNAMIC INACTIVATION OF RUBELLA VIRUS ENHANCES RECOMBINATION WITH A LATENT VIRUS OF A BABY HAMSTER KIDNEY CELL LINE BHK21*

Rubella virus is very sensitive to photodynamic action. When tested with 1.2 X 10(-5) M toluidine blue and 8 W fluorescent lamp at a fluence of 11 W/m2, inactivation kinetics showed a linear single hit curve with a k value of 1.48 min-1. Photodynamic inactivation of rubella virus greatly enhanced recombination with a latent virus (R-virus) of baby hamster kidney BHK21 cells. In contrast, no hybrids were detected in lysates of the cells infected with either UV-treated or untreated rubella virus. Therefore, hybrid viruses were readily detected only in lysates of BHK21 cells infected with photodynamically treated rubella virus. Photodynamic damage of rubella virus genomes generated a new hybrid type (hybrid type 3) in addition to a previously described type 2 hybrid (formerly designated as HPV-RV variant). Although both of these hybrid types carry the CF antigens of rubella virus, plaque forming ability of type 3 hybrid is neutralized neither by anti-rubella serum nor by anti-latent virus serum while type 2 hybrid is neutralized by anti-latent virus serum.     

REACTIVATION OF UV- AND γ-IRRADIATED HERPES VIRUS IN UV- AND X-IRRADIATEDCV–1 CELLS

Abstract— Enhanced reactivation of UV- and y-irradiated herpes virus was investigated by the plaque assay onCV–1 monkey kidney monolayer cells irradiated with UV light or X-rays. Both UV- and X-irradiatedCV–1 cells showed enhancement of survival of UV-irradiated virus, while little or no enhancement was detected for y-irradiated virus assayed on UV- or X-irradiated cells. The enhanced reactivation of UV-irradiated virus was greater when virus infection was delayed 24 or 48 h, than for infection immediately following the irradiation of cells. It is demonstrable that the UV- or X-irradiatedCV–1 cells are able to enhance the repair of UV damaged herpes virus DNA, but not of y-ray damaged ones.     

UV-ENHANCED VIRUS REACTIVATION IN MAMMALIAN CELLS: EFFECTS OF METABOLIC INHIBITORS*

Abstract—The induction process of UV-enhanced reactivation of UV-irradiated herpes simplex virus was investigated in CV-1 monkey kidney cells. A protein synthesis inhibitor, cycloheximide (0.5–5 μg/m/), present in the culture medium For 24 h between cell irradiation and virus infection decreased the enhanced virus survival normally found in UV-irradiated cultures. The enhanced virus reactivation became essentially resistant to the addition of cycloheximide by 6–8 h after cell irradiation, indicating that the cycloheximide-sensitive process necessary for enhanced reactivation was complete by that time. Since cycloheximide not only inhibits protein synthesis, but DNA synthesis as well, we investigated the effect of a DNA synthesis inhibitor, hydroxyurea. Hydroxyurea did not decrease UV-enhanced virus survival, but resulted in enhanced virus survival even in unirradiated cells. Therefore, the cycloheximide-caused inhibition of UV-enhanced reactivation did not arise from inhibition of DNA synthesis. The combined results indicate that (1) UV-enhanced virus reactivation in monkey kidney cells requires de novo protein synthesis during the first 6–8 h after cell irradiation and that (2) DNA synthesis inhibition may be the initiating event.     

PHOTODYNAMIC EFFECTS OF PROFLAVINE ON BACTERIOPHAGE φ times 174 AND ITS ISOLATED DNA

Abstract. Proflavine-mediated photoinactivation of φ times 174 phage and its isolated DNA was studied under identical irradiation conditions. The inactivations followed single-hit kinetics and a linear relationship was obtained in reciprocal plots of the inactivation rates vs the proflavine concentrations for both phage and isolated DNA. The phage photoinactivation rate was increased with an increase in the amount of proflavine bound to the phage DNA in a strong binding range (0.01-0.04 proflavine/ nucleotide) as the total proflavine concentration was increased or the ionic strength decreased. Further, a phage-specific factor was also found to affect the inactivation rate. The photodynamic treatment induced mutations in three phage strains from "amber" to "wild type" at a mutation rate per lethal hit of 0.3 times 10^-5 to 2.6 times 10^-5. In contrast to phage infectivity, the φ times 174 DNA infectivity was measurable only at a high multiplicity of infection, and its photoinactivation occurred only at high proflavine concentrations. The photoinactivation rate was enhanced either with a decrease in the multiplicity of infection or with the use of spheroplasts of recA mutants strains. The results are discussed in terms of the nature of and possible repair mechanisms of photodynamically induced lesions in φ times 174 phage DNA.     

Radiation enhanced reactivation of nuclear replicating mammalian viruses.

Abstract— When CV-1 monkey kidney cells were UV-irradiated (0–18 J/m²) or X-irradiated (0–10krads) before infection with UV-irradiated simian adenovirus 7 (SA7) or simian virus 40 (SV40), increases in the infectivity of these nuclear replicating viruses as measured by plaque formation were observed. These radiation enhanced reactivations, UV enhanced reactivation (UVER) and X-ray enhanced reactivation (X-ray ER), occurred both when virus infection immediately followed irradiation of the cells (except for X-ray ER with SA7) and when virus infection was delayed until 3–5 days after cell irradiation. While there was little difference in the levels of reactivation of UV-irradiated SV40 between immediate and delayed infection, delayed infection resulted in higher levels of reactivation of SA7. X-ray enhanced reactivation of UV-irradiated Herpes simplex virus persisted for several days but did not increase. Thus, X-ray enhanced and UV enhanced reactivations of these mammalian viruses were relatively long-lived effects. Essentially no UVER or X-ray ER was found in CV-1 cells for either immediate or delayed infection with UV-irradiated vaccinia virus or poliovirus, both of which replicate in the cell cytoplasm. These results suggest UVER and X-ray ER in mammalian cells may be restricted to viruses which are replicated in the cell nucleus.     

Synergistic Interaction of Photodynamic Treatment with the Sensitizer Aluminum Phthalocyanine and Hyperthermia on Loss of Clonogenicity of CHO Cells

Abstract— When CHO cells were exposed to hyperthermia and subsequently to photodynamic treatment, the combined effects were additive but in the reverse sequence the interaction was synergistic. The synergistic interaction comprised two quite different components: (1) photodynam-ically induced sensitization of cellular proteins and/or supramolecular structures for thermal inactivation and (2) a photodynamically induced inhibition of the cellular repair system for sublethal thermal damage. The first component of the synergistic interaction was reflected by a change of the Arrhenius parameters of thermal cell killing. A lowering of the activation energy of this process was responsible for the synergistic interactions, whereas a concomitant decrease of the frequency factor, opposing this effect, actually caused a much lower degree of synergism at higher temperatures. This component of the synergistic interaction did not respond to the insertion of an intermediate incubation period between the two treatments. The second component of the synergistic interaction, viz. the interference with the ability of cells to survive sublethal thermal damage, was reversible, as an intermediate incubation between photodynamic treatment and hyperthermia resulted in its repair. The photodynamically induced inhibition of the ability of cells to survive sublethal thermal damage was not related to ATP or glutathione depletion, inhibition of de novo protein synthesis or impairment of degradation of damaged protein molecules. Restoration of the repair system for sublethal damage depended on a metabolic process and required free intracellular Ca²⁺, suggesting that a cell signaling pathway may be involved. Thus, in a practical sense the magnitude of the synergistic interaction between photodynamic treatment and hyperthermia depends on the length of the interval between the two treatments and on the temperature and duration of the subsequent thermal treatment. This may have significant consequences for the development of clinical protocols.     

CELL DENSITY DEPENDENCE OF ULTRAVIOLET LIGHT ENHANCED REACTIVATION OF Herpes simplex TYPE I AND THE LARGE PLAQUE EFFECT IN C3H/10T1/2 MOUSE FIBROBLASTS

C3H/10T1/2 mouse fibroblasts were grown to different cell densities either by plating at low density and allowing different growth periods, or by plating at a series of increasing densities and allowing the same growth period. These plates were UV irradiated at 7.5 J/m2 or mock irradiated and 24 h later infected with UV-irradiated Herpes simplex type I virus which had been UV irradiated at 50 or 125 J/m2 or mock irradiated. The numbers and sizes of plaques were measured and these data used to calculate the extent of UV-enhanced host cell reactivation, the capacity enhancement, the large plaque effect (LPE) and the small plaque effect (SME). The influence of cell density on these phenomena was similar for both series of density experiments. Ultraviolet-enhanced host cell reactivation could be demonstrated only for cultures of lower density. The capacity of the cells for Herpes simplex type I virus decreased with cell density, but UV irradiated cells showed an increase in capacity with cell density. Plaque sizes decreased in all cases with cell density but the LPE and SPE were not significantly altered. The greatest variation in the above parameters occurred just as the cells were approaching confluence, where most host cell reactivation experiments are carried out. We conclude that the reproducibility of such experiments depends critically on cell density, a dependence which may be relevant to mechanistic interpretations of the UV-dependent phenomena.     

THE EFFECT OF METABOLIC INHIBITORS ON THE LARGE PLAQUE EFFECT WITH HERPES SIMPLEX VIRUS

Abstract— Herpes simplex virus (HSV) macroplaque strain plaque development is faster on ultraviolet (UV)-irradiated African green monkey kidney cells if viral infection is delayed for 12 h or more after cell irradiation (Coohill et al. , 1980). This phenomenon has been termed the large plaque effect (LPE). Here we show that treatment of UV-irradiated cells with cycloheximide inhibits the LPE. Pretreatment of unirradiated cells with hydroxyurea, caffeine, or acetoxy-acetylaminofluorene results in faster plaque development. Treatment of UV-irradiated cells with either hydroxyurea or caffeine gave a LPE of the same magnitude as UV alone. In addition, the LPE was observed with other HSV strains—microplaque, syn-20, and KOS. Our results are consistent with the interpretation that the LPE is 'inducible' in African green monkey kidney cells and that inhibition of DNA synthesis is the inducing event. Possible causes of the LPE and similarities between the LPE and enhanced viral reactivation are discussed.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET INACTIVATION OF HOST CAPACITY IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract. The ability of UV-irradiated African green monkey kidney cells (CV-1) to support the growth of unirradiated herpes simplex virus type 1 as measured by plaque forming ability has been investigated. The lowering of plaque formation by the virus when the host cell was irradiated was examined at thirteen different wavelengths. An action spectrum for this cellular parameter (capacity) was obtained in the wavelength region of 235–302 nm. This action spectrum points to nucleic acid as the critical target molecule for this effect.     

THE EFFECTS OF ULTRAVIOLET LIGHT ON HOST CELL REACTIVATION AND PLAQUE SIZE OF Herpes simplex VIRUS TYPE I IN C3H/lOT1/2 MOUSE CELLS

— Herpes simplex virus — type 1 (HSV-I) plaque-forming ability and plaque size were measured on C3H/1OT1/2 cell monolayers as functions of pretreatment dose with UV light at different times before inoculation with virus, in order to determine if UV-enhanced reactivation (ER) of UV-irradiated virus. as well as associated phenomena, could be obtained in this cell system. The number of virus plaques observed (i.e. the capacity of the cells to support virus growth) and the size of the plaques were found to increase substantially with pretreatment of the cells with UV light. However, no significant ER was observed. Therefore, the mechanisms responsible for the increases in plaque size and cell capacity seem to be independent of those responsible for ER. In work by others. C3H/l0T1/2 cells have hcen transformed by UV light at doses similar to those used in this study; the absence of ER of UV-irradiated virus in this study indicates that the mechanism underlying ER is not required for transformation.     

THE WAVELENGTH DEPENDENCE OF 8-METHOXYPSORALEN PHOTOSENSITIZATION OF HOST CAPACITY INACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract— The addition of 8-methoxypsoralen to cultures of African green monkey cells (CV-I) sensitized the inactivation by near UV radiation (302–370 nm) of the ability of the cells to host herpes simplex virus. No sensitizing effect by drug addition was noted for far UV radiation (232–297 nm). An action spectrum for the photosensitized inactivation of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum of 8-methoxypsoralen.     

UV-ENHANCED REACTIVATION IN MAMMALIAN CELLS: INCREASE BY CAFFEINE

Abstract— It has been reported that caffeine decreases UV-enhanced reactivation of UV-irradiated Herpes simplex virus in CV-1 monkey kidney cells. That occurred when there was no delay between cell irradiation and virus infection. In the present study, virus infection was delayed following cell irradiation to allow an 'induction'period separate from the 'expression'period which occurs during the virus infection. Thus, the effects of caffeine on 'induction'and 'expression'could be determined separately. Caffeine increased the expression of UV-enhanced reactivation, while causing a small decrease in the 'induction'of enhanced reactivation.     

Activation of macrophage tumoricidal activity by photodynamic treatment in vitro-indirect activation of macrophages by photodynamically killed tumor cells

Macrophages constitute a major part of natural tumor defense by their capacity to destroy selectively a broad range of tumor types upon specific activation. In the last couple of years, these cells have also been implicated as effector cells in the destruction of tumors by photodynamic therapy. In the present work, the potential role of macrophage-mediated tumor cytotoxicity after photodynamic treatment in vitro has been investigated with respect to photodynamic activation of macrophages for tumoricidal effector functions. Our data show that photodynamic treatment of highly pure murine bone-marrow-derived macrophages with the hematoporphyrin derivative Photosan-3 does not result in activation of these cells for cytotoxicity against YAC-1 tumor cells or secretion of tumor necrosis factor and nitric oxide, irrespective of co-stimulation with interferon-γ, a potent priming agent for macrophage antitumoral activity. On the contrary, treatment with higher photosensitizer doses is found to reduce markedly the viability of the macrophage effector cells. Thus, these results do not lend any support to the hypothesis of direct macrophage activation by photodynamic treatment. However, macrophages are found to be activated for tumoricidal effector functions indirectly by photodynamically killed tumor cells, in a way reminiscent of phagocytosis-inducing stimuli. It is thus suggested that recognition and phagocytosis of photodynamically destroyed tumor cells constitutes the major signal for local activation of macrophages in photodynamically treated tumor tissues, which may be crucial for final, specific eradication by the immune system of tumor cells surviving photodynamic treatment.     

HOST CELL REACTIVATION AND UV-ENHANCED REACTIVATION IN SYNCHRONIZED MAMMALIAN CELLS

Abstract— Does host cell reactivation (HCR) or UV-enhanced reactivation (UVER) of UV-irradiated Herpes simplex virus (UV-HSV) vary during the host mammalian cell cycle? The answer could be useful for interpreting UVER and/or the two-component nature of the UV-HSV survival curve. Procedures were developed for infection of mitotically-synchronized CV-1 monkey kidney cells. All virus survival curves determined at different cell cycle stages had two components with similar D₀'s and intercepts of the second components. Thus, no single stage of the host cell cycle was responsible for the second component of the virus survival curve. When the cells were UV-irradiated immediately prior to infection, enhanced survival of UV-HSV occurred for cell irradiation and virus infection initiated during late G₁/early S phase or late S/early G₂ phase but not during early G₁ phase. For infection delayed by 24 h after cell irradiation, UVER was found at all investigated times. These results indicate that: (1) HCR is similar at all stages of the host cell cycle; and (2) the “induction” of UVER is not as rapid for cell-irradiation in early G₁ phase. This latter observation may be one reason why normal, contact-inhibited cells do not express UVER as rapidly as faster growing, less contact-inhibited cells.     

Sub-lethal Photodynamic Damage to ARPE-19 Cells Transiently Inhibits Their Phagocytic Activity†

Efficient phagocytosis of photoreceptor outer segments (POS) membranes by retinal pigment epithelium (RPE) plays a key role in biological renewal of these highly peroxidizable structures. Here, we tested whether photodynamic treatment, mediated by merocyanine 540 (MC 540), rose Bengal or a zinc-substituted chlorophyllide inhibited phagocytic activity of ARPE-19 cells in vitro. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS isolated from cow retinas and nonspecific phagocytosis of fluorescent polystyrene beads were measured by flow cytometry. Photodynamic treatment, mediated by all three photosensitizers with sub-threshold doses, induced significant inhibition of the cell-specific phagocytosis. The nonspecific phagocytosis was inhibited by photodynamic treatment mediated only by MC 540. The inhibition of phagocytosis was a reversible phenomenon and after 24 h, the photodynamically treated cells exhibited phagocytic activity that was comparable with that of untreated cells. This study provides proof of principle that sub-threshold photodynamic treatment of ARPE-19 cells with appropriate photosensitizers is a convenient experimental approach for in vitro study of the effects of oxidative stress on specific phagocytic activity of RPE cells. We postulate that oxidative damage to key components of the cell phagocytic machinery may be responsible for severe impairment of its activity, which can lead to retinal degeneration.     

THE EFFECTS OF ULTRAVIOLET RADIATION EXPOSURE OF ADJACENT CELLS ON PLAQUE FORMATION WITH Herpes simplex VIRUS TYPE I

African green monkey kidney cells (CV-1P) were exposed to low fluences of 254 nm germicidal radiation and then infected with Herpes simplex virus, type I. The result of this treatment was an increase in viral plaque development rate, the large plaque effect (LPE). A measurement of the kinetics of plaque development suggested that a large portion of the effect could be due to events occurring in those cells that are adjacent to the initially infected cell. An infectious center assay was employed in order to isolate the effects of ultraviolet radiation (UV) on the initially infected cells from those effects on the adjacent cells that became infected as the plaque spread radially outward. Plaque development began earlier in UV irradiated cells and progressed at a uniformly accelerated rate compared to untreated cells. Results indicate that although the initially infected cell contributes to the LPE, the major effect is due to events that occur in the adjacent cells. Each round of viral replication appears to contribute equally to the LPE. The virally induced rate of fusion of the initially infected cell with its immediate neighbors is not affected by UV.     

The use of FTIR microscopy for evaluation of herpes viruses infection development kinetics

The kinetics of Herpes simplex infection development was studied using an FTIR microscopy (FTIR-M) method. The family of herpes viruses includes several members like H. simplex types I and II (HSV I, II), Varicella zoster (VZV) viruses which are involved in various human and animal infections of different parts of the body. In our previous study, we found significant spectral differences between normal uninfected cells in cultures and cells infected with herpes viruses at early stages of the infection. In the present study, cells in cultures were infected with either HSV-I or VZV and at various times post-infection they were examined either by optical microscopy or by advanced FTIR-M. Spectroscopic measurements show a consistent decrease in the intensity of the carbohydrate peak in correlation with the viral infection development, observed by optical microscopy. This decrease in cellular carbohydrate level was used as indicator for herpes viruses infection kinetics. This parameter could be used as a basis for applying a spectroscopic method for the evaluation of herpes virus infection development. Our results show also that the development kinetics of viral infection has an exponential character for these viruses.     

UV-ENHANCED REACTIVATION OF MINUTE-VIRUS-OF-MICE: STIMULATION OF A LATE STEP IN THE VIRAL LIFE CYCLE

Abstract— UV-enhanced reactivation of minute-virus-of-mice (MVM), an autonomous parvovirus, was studied in parasynchronous mouse A9 cells. The survival of UV-irradiated MVM is increased in cells which have been UV-irradiated prior to infection. UV-enhanced reactivation can be explained neither by facilitated plaque detection on UV-treated indicator cells, nor by altered kinetics of virus production by UV-irradiated cells. No effect of the multiplicity of infection on virus survival was detected in unirra-diated or irradiated cells. The magnitude of UV-enhanced reactivation is a direct exponential function of the UV dose administered to the virus while virus survival is inversely proportional to the UV dosage. The expression of UV-enhanced reactivation can be activated in cells arrested in G₀, it requires de novo protein synthesis and it is maximal when cells are irradiated 30 h before the onset of viral DNA replication. Early phases of the viral cycle, such as adsorption to cellular receptors, migration to the nucleus and uncoating, were not affected by cell irradiation and are unlikely targets of the UV-enhanced reactivation function(s). These results, together with the single-strandedness of the viral genome, strongly suggest that the step stimulated in UV-irradiated cells functions concomitant with, or subsequent to, viral DNA replication.     

Oligolysine‐Conjugated Zinc(II) Phthalocyanines as Efficient Photosensitizers for Antimicrobial Photodynamic Therapy

A series of zinc(II) phthalocyanines conjugated with an oligolysine chain (n=2, 4, and 8) were synthesized and characterized by using various spectroscopic methods. As shown by using UV/Vis and fluorescence spectroscopic methods, these compounds were nonaggregated in N,N‐dimethylformamide, and gave a weak fluorescence emission and high singlet oxygen quantum yield (Φ_Δ=0.86–0.89) as a result of their di‐α‐substitution. They became slightly aggregated in water with 0.05 % Cremophor EL, but they could still generate singlet oxygen effectively. The antimicrobial photodynamic activities of these compounds were then examined against various bacterial strains, including the Gram‐positive methicillin‐sensitive Staphylococcus aureus ATCC 25923 and methicillin‐resistant Staphylococcus aureus ATCC BAA‐43, and the Gram‐negative Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853. Generally, the dyes were much more potent toward the Gram‐positive bacteria. Only 15 to 90 nM of these photosensitizers was required to induce a 4 log reduction in the cell viability of the strains. For Escherichia coli, the photocytotoxicity increased with the length of the oligolysine chain. The octalysine derivative showed the highest potency with a 4 log reduction concentration of 0.8 μM . Pseudomonas aeruginosa was most resistant to the photodynamic treatment. The potency of the tetralysine derivative toward a series of clinical strains of Staphylococcus aureus was also examined and found to be comparable with that toward the nonclinical counterparts. Moreover, the efficacy of these compounds in photodynamic inactivation of viruses was also examined. They were highly photocytotoxic against the enveloped viruses influenza A virus (H1N1) and herpes simplex virus type 1 (HSV1), but exhibited no significant cytotoxicity against the nonenveloped viruses adenovirus type 3 (Ad3) or coxsackievirus (Cox B1). The octalysine derivative also showed the highest potency with an IC₅₀ value of 0.05 nM for the two enveloped viruses.     

THE EFFECT OF PHOTODYNAMIC TREATMENT OF YEAST WITH THE SENSITIZER CHLOROALUMINUM PHTHALOCYANINE ON VARIOUS CELLULAR PARAMETERS

Photodynamic treatment of Kluyveromyces marxianus with chloroaluminum-phthalocyanine resulted in loss of clonogenicity. Several parameters were studied to identify targets that could be related to loss of colony-forming capacity. Inhibition of various plasma membrane-bound processes was observed, such as substrate transport and plasma membrane ATPase activity. Moreover, K+ loss from the cells was observed. Photodynamic treatment also reduced the activity of various enzymes involved in energy metabolism, thereby decreasing the cellular ATP level. It will be discussed however that none of these processes is likely to be related directly to loss of clonogenicity. Treatment with phthalocyanine and light resulted in a strong inhibition of the incorporation of 14C-phenylalanine in trichloracetic acid-precipitable material. The induction of the β-galactoside utilization system was also strongly inhibited. The latter two processes did not recover during incubation, subsequent to photodynamic treatment. It is concluded that photodynamically induced inhibition of protein synthesis is a critical factor contributing to the loss of clonogenicity.