Oxygen-dependent laser inactivation of murine norovirus using visible light lasers.

BACKGROUND: Previous work indicated that an ultrashort pulse (USP) 425 nm laser is capable of inactivating murine norovirus (MNV: Virol. J. 11:20), perhaps via an impulsive stimulated Raman scattering (ISRS) mechanism, and does not substantially damage human plasma proteins (PLOS One 9:11). Here, further investigation of virus inactivation by laser light is performed. METHODS: In this study, we evaluate whether inactivation of MNV is specific to the USP wavelength of 425 nm, or if it occurs at other visible wavelengths, using a tunable mode-locked Ti-Sapphire laser that has been frequency doubled to generate femtosecond pulses at wavelengths of 400, 408, 425, 450, 465, and 510 nm. Continuous Wave (CW) lasers are also applied. Singlet oxygen enhancers are used to evaluate the sensitivity of MNV to singlet oxygen and oxygen quenchers are used to evaluate effects on virus inactivation as compared to untreated controls. RESULTS: > 3 log10 inactivation of MNV pfu occurs after irradiation with an average power of 150 mW at wavelengths of 408, 425 or 450 nm femtosecond-pulsed light for 3 h. Thus results suggest that the mechanism by which a laser inactivates the virus is not wavelength-specific. Furthermore, we also show that irradiation using a continuous wave (CW) laser of similar power at 408 nm also yields substantial MNV inactivation indicating that inactivation does not require a USP. Use of photosensitizers, riboflavin, rose bengal and methylene blue that generate singlet oxygen substantially improves the efficiency of the inactivation. The results indicate a photochemical mechanism of the laser-induced inactivation where the action of relatively low power blue laser light generates singlet oxygen. CONCLUSION: Results suggest formation of short-lived reactive oxygen species such as singlet oxygen by visible laser light as the cause of virus inactivation rather than via an ISRS mechanism which induces resonant vibrations.

Evaluation of the inactivation of Venezuelan equine encephalitis virus by several common methods.

Working with virological samples requires validated inactivation protocols for safe handling and disposal. Although many techniques exist to inactivate samples containing viruses, not all procedures have been properly validated or are compatible with subsequent assays. To aid in the development of inactivation protocols for Alphaviruses, and specifically Venezuelan equine encephalitis virus (VEEV), a variety of methods were evaluated for their ability to completely inactivate a high titer sample of the vaccine strain VEEV TC-83. The methods evaluated include reagents used in RNA extraction, fixation, treatment with a detergent, and heat inactivation. Most methods were successful at inactivating the sample; however, treatment with only Buffer AVL, SDS, and heat inactivation at 58 °C for one hour were not capable of complete inactivation of the virus in the sample. These results provide a substantial framework for identifying techniques that are safe for complete inactivation of Alphaviruses and to advise protocol implementation.

Temperature and density dependent induction of a cytopathic effect following infection with non-cytopathic HAV strains.

Hepatitis A virus infection and growth in cultured cells is protracted, cell-type restricted, and generally not accompanied by the appearance of a cytopathic effect, with the exception of some culture-adapted strains. We demonstrate that the non-cytopathic HAV strain HM175/clone 1 can be induced to exhibit a cytopathic phenotype in both persistently or acutely infected cells under co-dependent conditions of lower incubation temperature (<34°C) and reduced cell density in both monkey (FRhK-4) and human (A549) cells. This phenotype is not virus-strain restricted, as it was also observed in cells infected with HAV strains, HAS-15 and LSH/S. Cytopathic effect was accompanied by rRNA cleavage, indicating activation of the RNase L pathway, viral negative strand synthesis, caspase-3 activation, and apoptosis. The results indicate that a cytopathic phenotype may be present in some HAV strains that can be induced under appropriate conditions, suggesting the potential for development of a plaque assay for this virus.

Gene expression and cytopathic effect of vaccinia virus inactivated by psoralen and long-wave UV light.

Induction of the cytopathic effect (CPE) in cells infected with poxvirus seems ubiquitous in that it has been associated with all different strains and preparations of poxviruses, regardless of the replicating status of these viruses. The study of the mechanisms by which CPE is induced by nonreplicating poxviruses is hampered by the lack of any noncytopathic mutant strains and preparations. In this paper, we report on the patterns of gene expression and induction of CPE by vaccinia viruses treated by limited cross-linking with psoralen and long-wave UV light (PLWUV). We show that treatment of cell-free virus with PLWUV could inactivate viral replication without abolishing the ability of the virus to infect cells. Viral transcription as indicated by reporter genes was generally enhanced and prolonged under early viral promoters and abolished under late promoters. Furthermore, increasing the levels of cross-linking with PLWUV resulted in a decrease and abolishment of viral expression of a large reporter gene and a concomitant loss of the induction of CPE. Cells infected with such a virus were able to express the reporter genes and proliferate. The generation of nonreplicating and noncytopathic recombinant vaccinia viruses may help in studies of the mechanisms of CPE induction by poxvirus and may facilitate the use of poxviral vectors in broader areas of research and clinical applications.

Control of baculovirus gp64-induced syncytium formation by membrane lipid composition.

We have investigated the effects of membrane lipid composition on biological membrane fusion triggered by low pH and mediated by the baculovirus envelope glycoprotein gp64. Lysolipids, either added exogenously or produced in situ by phospholipase A2 treatment of cell membranes, reversibly inhibited syncytium formation. Lysolipids also decreased the baculovirus infection rate. In contrast, oleic and arachidonic acids and monoolein promoted cell-cell fusion. Membrane lipid composition affected pH-independent processes which followed the low-pH-induced change in fusion protein conformation. Inhibition and promotion of membrane fusion by a number of lipids could not be explained by mere binding or incorporation into membranes, but rather was correlated with the effective molecular shape of exogenous lipids. Our data are consistent with the hypothesis that membrane fusion proceeds through highly bent membrane intermediates (stalks) having a net negative curvature. Consequently, inverted cone-shaped lysolipids inhibit and cone-shaped cis-unsaturated fatty acids promote stalk formation and, ultimately, membrane fusion.

The anti-HIV activity of the phytochemical alpha-terthienyl.

The plant trithiophene, alpha-terthienyl (alpha T), was evaluated for activity against the human immunodeficiency virus (HIV-1). Antiviral activity specifically required long wavelength light (UVA, 320-400 nm). The compound had little or no activity in visible light or in the dark. The anti-HIV effect was UVA-dose dependent and was proportional to the concentration of alpha T, according to several parameters of virus infectivity and replication. The efficacy was decreased to some extent by the presence of bovine serum in the reactions; but under optimal conditions 0.1 microgram/ml. alpha T (3 x 10(-7) M) could inactivate 10(4)-10(5) infectious particles. In contrast poliovirus and Coxsackievirus infectivity were relatively resistant to alpha T + UVA.

Photoinactivation of viruses in human fresh plasma by phenothiazine dyes in combination with visible light.

We developed a photodynamic method to inactivate viruses in human fresh plasma. Single plasma bags were illuminated with visible light in the presence of low doses of phenothiazine dyes like methylene blue or toluidine blue. By this treatment the infectivity of different enveloped viruses including the causative agent of AIDS, HIV-1, was completely removable from the plasma. Non enveloped viruses, however, proved to be more stable. The activities of clotting factors and other plasma proteins were only slightly decreased. There was no indication that the procedure led to important structural modifications of plasma proteins. The dyes are photodynamically active at concentrations much lower than those at which they are therapeutically used as antidots in the treatment of methemoglobinemia.

[The effect of infrared laser rays on herpes simplex virus and the functions of human immunocompetent cells]. / Einfluss infraroter Laserstrahlen auf das Herpes-simplex-Virus und Funktionen menschlicher immunkompetenter Zellen.

In vitro investigations were performed to study the effect of infrared Nd:YAG laser irradiation on herpes simplex virus and the viral replication in herpes-infected Vero cell microcultures. In addition, the influence of laser irradiation on human immuncompetent cells was investigated by irradiation and incubation of herpes-infected cell cultures overlaid with leucocytes and observation of leucocyte migration under agarose after laser irradiation. There was no evidence of herpes simplex virus inactivation by laser irradiation. Irradiation of Vero cell microcultures infected with virus did not influence the development of the viral cytopathic effect. However, irradiated cultures showed an increase of about 50% in the virus yield. Only a slight indication of laser influence on immunocompetent cells was found. Cell cultures incubated with leucocytes (from both seropositive and seronegative donors) and irradiated showed an inhibitory effect with about half the virus yield seen in unirradiated controls. However, there was no relation between the energy applied and yield reduction. In addition, laser light caused no change in leucocyte migration.

Quantitative evaluations of the effect of UV irradiation on the infectivity of HTLV-III (AIDS virus) with HTLV-I-carrying cell line, MT-4.

The effect of UV irradiation on HTLV-III was quantitatively studied to evaluate the dosage of UV irradiation which inactivates the virus for sterilization of blood products and for laboratory decontamination. In order to estimate the biologic activity and quantitation of the virus, induction of HTLV-III-specific antigens and inhibition of DNA synthesis in MT-4 cells infected by UV-irradiated HTLV-III were detected by indirect immunofluorescence technique and proliferation assay using [3H]thymidine uptake, respectively. Furthermore, plaque-forming assay was performed to count the infectious viral particles. Results showed that HTLV-III was completely inactivated by 5000 J/m2 UV irradiation. Cloned UV-irradiated HTLV-III (UV-1) was obtained from a plaque that was formed by 2000 J/m2 UV-irradiated virus. When MT-4 cells were infected by the clone UV-1, ballooning degeneration of cells was predominantly induced. These ballooning cells were not usually observed in MT-4 cells infected by unirradiated HTLV-III. The resistance to UV was not different between clone UV-1 and unirradiated HTLV-III.

Production of cytoplasmic polyhedrosis virus (CPV) polyhedra in a gamma irradiated Lymantria dispar cell line.

Lymantria dispar cells wee exposed to different doses of gamma radiation one hour after infection with cytoplasmic polyhedrosis virus (CPV). It was found that irradiated cells can produce infectious polyhedra. Modifications in the structure and in the process of maturation of the polyhedra were noted. The number of polyhedra per cell increased significantly after cell irradiation at 10(4) and 10(5) rads but no change was noted after cell treatment at 10(2) rads. On the other hand, morphological changes and a high mortality rate were noted in cell cultures treated at intensities higher than 10(3) rads. Therefore, the total yield of polyhedra produced when using 10(2) or 10(4) rads was similar to that obtained in normal cells but dropped significantly after cell irradiation at 10(5) rads.

[Conditions controlling the development of distant intercellular interactions during ultraviolet radiation]. / Usloviia vozniknoveniia distantnykh mezhkletochnykh vzaimodeistvii pri UF-radiatsii.

The study is concerned with ascertaining the role of UV radiation in distant intercellular interactions (DII) and the conditions resulting in "MIRROR" CYTOPATHIC EFFECT ("M" CPE). It has been found that the UV radiation-induced extreme state of the cells in a radiant culture produces distantly in an intact detector culture, which has only an optic contact with it, the cytopathic effect (CPE) as a repercussion of a specificity of morphological manifestations imprinted in the affected culture. UV preparadiation of the detector cells aids in manifestation of the "mirror" CPE.

The effects of ultraviolet and ionizing radiation on herpesviruses, SV40 and adenoviruses in relation to the small-plaque effect.

The small-plaque effect occurs with a wide range of herpesviruses following irradiation with ultraviolet light. The 37 per cent survival (D37) values, or dose required for one lethal hit (e-1), for herpes simplex, pseudorabies and pigeon herpesviruses in different cells indicate a broad spectrum of host-cell repair capacity. Other DNA-containing viruses such as SV40 and adenoviruses, which also replicate in the cell nucleus, show the small-plaque effect. Ionizing irradiation of herpes simplex virus type 1 (HSV-1) showed but little reduction in plaque-size.

Effect of X rays on virus production and DNA synthesis in adenovirus-infected cells.

The effect of X rays on the process of type 10 adenovirus infection in HeLa cells was tested. CPE development and virus infections titer were lower in the irradiated cells, while CF titer remained unchanged. The rate of DNA synthesis, as measured by 3H-thymidine incorporation, was higher in the infected cells and radiosensitivity of the process was higher in the infected cells compared with uninfected controls.

Interaction of encephalomyocarditis virus with ultraviolet-irradiated host cells.

Ultraviolet light (UV) impaired the capacity of L cells to support growth of encephalomyocarditis virus. The loss of capacity was partially restored by high multiplicity of infection (MOI). This phenomenon was not due to an increased probability of an infectious virus particle reaching a site of replication undamaged by UV, since UV-inactivated virus at high MOI induced restoration of the capacity to support multiplication of nonirradiated virus adsorbed at low MOI. Multiplicity reactivation of UV-irradiated virus did not play a role in this phenomenon since restoration of capacity took place without multiplication of the UV-irradiated restoring virus. The evidence indicates that restoration of capacity was not due to viral interactions involving genetic exchange. The ability to restore capacity was a property more radioresistant than infectivity, suggesting that the former is a function only of part of the viral genome.