DNA Damage Kills Bacterial Spores and Cells Exposed to 222-Nanometer UV Radiation.

This study examined the microbicidal activity of 222-nm UV radiation (UV222), which is potentially a safer alternative to the 254-nm UV radiation (UV254) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, and Clostridioides difficile and a herpesvirus were all killed or inactivated by UV222 and at lower fluences than with UV254B. subtilis spores and cells lacking the major DNA repair protein RecA were more sensitive to UV222, as were spores lacking their DNA-protective proteins, the α/ß-type small, acid-soluble spore proteins. The spore cores' large amount of Ca2+-dipicolinic acid (∼25% of the core dry weight) also protected B. subtilis and C. difficile spores against UV222, while spores' proteinaceous coat may have given some slight protection against UV222 Survivors among B. subtilis spores treated with UV222 acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV222 resistance. UV222-treated B. subtilis spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV222 does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV222IMPORTANCE Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of Bacillus anthracis Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV254) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV222) may be less harmful to people than UV254 yet may still kill bacteria and at lower fluences than UV254 The present work has identified the damage by UV222 that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV222 also inactivates a herpesvirus.

A case of herpes simplex virus reactivation after fractional ablative carbon dioxide laser to treat a burn scar.

Fractional photothermolysis was initially introduced by Manstein in 2004 .Fractional CO2 laser technology introduced has allowed physicians to obtain good cosmetic results with a lower rate of complications than non-fractionated ablative laser treatment. However, adverse effects may still occur.Reported cases of HSV infection after fractional photothermolysis are rare. A 48-year-old woman with Fitzpatrick skin type III presented with a scar in her perioral area desiring esthetic improvement of her burn scar. She didn't have a history of recurrent herpes simplex virus (HSV) infection periorally. A fractionated resurfacing laser Quadralase (Candela) was used to treat her perioral burn scar. Two sessions were performed with a month interval. Five days after the second session of laser therapy even after she took antiviral prophylaxis based on valacyclovir 500mg twice daily 24 hours before the laser session and 3 days after, she presented with a rash on the perioral area preceded by pain. Correlation of the history and the clinical presentation was consistent with HSV reactivation. Treatment was initiated with acyclovir 10mg/kg/8h administered intravenously for 10 days with a clearing of her vesicular eruption. Fractional CO2 laser is a very safe procedure when used with accepted parameters. Early recognition, close monitoring and careful wound care will prevent long term sequelae when complications occur.

Immunologic manipulation of metastases due to herpesvirus transformed cells.

Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and simian virus 40 (SV40) fail to induce immunity in weanling Syrian hamsters to transplant of hamster cells transformed by HSV-2. However, the development of metastatic tumors is markedly enhanced by prior immunization with HSV-1. Immunization with SV40, ultraviolet-irradiated tumor cells, or ultraviolet-irradiated normal hamster embryo cells inhibits the development of metastases. The HSV-hamster system appears a good one for the study of development, prevention, and control of metastases by mammalian cells transformed by a common human virus.

Mechanisms of photodynamic inactivation of herpes simplex viruses: comparison between methylene blue, light plus electricity, and hematoporhyrin plus light.

Herpes simplex virus (HSV) types 1 and 2 have been inactivated in vitro using low concentrations of methylene blue (MB), light (lambda) plus electricity (E), or hematoporphyrin derivative (HPD) plus lambda. Both techniques introduce single strand interruptions into viral DNA, but do not make double strand ruptions into viral DNA, but do not make double strand breaks. MB, lambda plus E-treated virions adsorb normally to and penetrate susceptible cells, whereas HSV inactivated with HPC and light does not. This difference is emphasized by the induction of new viral and cell DNA synthesis after infection with MB, lambda plus E-treated virions, whereas only cell, DNA but no HSV DNA, is made subsequent to HPD and lambda exposure. These observations reflect disparate mechanisms of viral inactivation. A block(s) in viral maturation, subsequent to viral DNA synthesis, occurs as a result of treatment with MB, lambda and E, whereas HPD plus lambda-treated particles fail to enter a susceptible cell, and therefore do not initiate an infection.

Enhanced reactivation and enhanced mutagenesis of herpes simplex virus in normal human and xeroderma pigmentosum cells.

Enhanced reactivation (ER) and enhanced mutagenesis (EM) of herpes simplex virus type 1 were studied simultaneously in UV-irradiated stationary cultures of diploid normal human and xeroderma pigmentosum (XP) fibroblasts. Mutagenesis was assayed with unirradiated herpes simplex virus type 1 as a probe in a forward mutation assay (resistance to iododeoxycytidine). Dose-response studies showed that ER increased with the UV dose given to the virus. Optimal reactivation levels were obtained when normal cells and XP variant cells were exposed to a UV dose of 8 J . m-2 and the virus was irradiated with 150 J . m-2. Repair-deficient XP cells of complementation groups A, C, and D showed optimal reactivation levels with a UV dose to the cells of 1.0 J . m-2 and a UV dose to the virus of 40 J . m-2. The time course of appearance of ER and EM was also studied, both in the normal and XP cells. In all cell types except the XP variant cells, EM followed similar kinetics of appearance as did ER. Maximal activities occurred when infection was delayed 1 or 2 days after cell treatment. In XP variant cells, however, maximal expression of the EM function was significantly delayed with respect to ER. The results indicate that ER and EM are transiently expressed in normal and repair-deficient XP cells. Although both phenomena may be triggered by the same cellular event, ER and EM appear to be separate processes that occur independently of each other.

Chromosome aberrations in Syrian hamster embryo cells transformed after exposure to ultraviolet-irradiated herpes simplex virus type 1 or 2.

Six Syrian hamster embryo cell lines (14-012-8-1, KOS-6-1, 333-8-9, 333-2-29, MS-4-1, FR-6-1), developed after exposure of primary cultures to different strains of UV-irradiated herpes simplex virus (HSV) type 1 or 2, were analyzed for chromosome aberrations. All the cell lines showed chromosome stability (number of chromosomes were maintained within a narrow range of variation in the diploid region) and a low incidence of polyploids, endoreduplications, and metaphases with pulveration or extensively fragmented chromosomes. The cell lines, passaged over long periods of time in vitro, developed marker chromosomes that suggested a clonal-type evolution of the cell populations. Two cell lines, 333-8-9 and 14-012-8-1, showed two different marker chromosomes with large heterochromatic regions. Chromosomes with abnormal heterochromatic regions, which often appeared like prominent secondary constrictions, were found in all the cell lines we examined. The level of chromosome breakage was low in all the cell lines except the highly tumorigenic cell line 333-2-29, which had a high incidence of cells with single or double chromatinic bodies. The abnormal heterochromatic regions that occurred on marker chromosomes and prominent secondary constrictions were interpreted as a possible chromosomal effect of the HSV. The karyotypic stability and low incidence of open breaks might have been the result of UV irradiation of the HSV.

Defective reactivation of ultraviolet light-irradiated herpesvirus by a Bloom's syndrome fibroblast strain.

We have used the technique of host cell reactivation of UV-irradiated herpes simplex virus type 1 as a measure of the repair capacity of three Bloom's syndrome skin fibroblast strains. At low multiplicity of infection (less than 6 x 10(-4) plaque-forming unit/cell), reactivation of the virus by the Bloom's syndrome strains was indistinguishable from that by normal strains. Reactivation at higher multiplicities was measured using an infectious centers assay. At 3 plaque-forming units/cell, survival of UV-irradiated herpes simplex virus was higher in all cell strains as a result of the multiplicity reactivation effect. This effect was, however, much smaller in one Bloom's syndrome strain, GM1492, than in either the normal strains or the other Bloom's syndrome fibroblasts. The defect in GM1492 was manifest only at relatively high multiplicates of infection. Thus, at 0.01 plaque-forming unit/cell, the GM1492 strain appeared normal, using the infectious centers assay. Clonal survival of the UV-irradiated GM1492 fibroblasts was also normal. Caffeine at 4 mM had little effect on either virus or cell survival following UV irradiation. The results indicate that the Bloom's syndrome strain GM1492 may be deficient in one of the cellular functions responsible for the multiplicity reactivation effect. These effects include complementation and recombinational events. Alternatively, the GM1492 strain may have a defective UV repair system which becomes saturated at high levels of damage.

Quantitative differences in host cell reactivation of ultraviolet-damaged virus in human skin fibroblasts and epidermal keratinocytes cultured from the same foreskin biopsy.

Repair efficiency of cultured cells may be estimated by measuring the ability of a particular cell type to support virus damaged by an appropriate agent. In this study we have compared the inactivation of ultraviolet (254 nm)-damaged herpes simplex virus in human fibroblast and epidermal keratinocyte cell lines derived from the same foreskin biopsy and found the epithelial cells to be a factor of 3 times less efficient in supporting the damaged virus. The two different cell types show comparable ultraviolet inactivation of clone-forming ability, indicating that the difference is specific to viral host cell reactivation. This study required the development of a quantitative infectious centers assay for the measurement of viral titer in human epithelial cells, a system which may be of more general application in studies of potential human carcinogens.

Light-induced mutagenicity of neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride).

Illumination of Salmonella typhimurium in the presence of neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) results in mutations of the base substitution type.

Repair of DNA following incorporation of 1-beta-D-arabinofuranosylcytosine into herpes simplex virus type 1.

The nucleoside analogue 1-beta-D-arabinofuranosylcytosine (ara-C) is incorporated into herpes simplex virus type 1 (HSV-1) DNA, and this correlates with inhibition of virus replication. The technique of Weigle-type reactivation (WR) was used to compare the ability of induced cellular DNA repair pathways to recognize or repair ara-C incorporated into HSV-1 DNA and ultraviolet (UV)-irradiated virus DNA (254 nm). Pretreatment of monkey cells with low-fluence UV irradiation, growth in cis-dichlorodiammineplatinum(II), or growth in ara-C followed by infection after a 24-hr incubation period resulted in enhanced survival of UV-irradiated HSV-1. Under the same experimental conditions, no reactivation of HSV-1 inactivated by growth in ara-C is observed. Comparisons between uninfected Vero cells exposed to UV irradiation (30 J/m2) or grown in 10(-6) M ara-C demonstrated repair replication in irradiated cells, whereas there was no evidence for DNA repair at various time intervals following removal of the nucleoside analogue. These observations suggest that, once ara-C is incorporated into HSV-1 or eukaryotic DNA, it is not recognized as a repairable lesion within the limits of the DNA repair assays used in these studies.

Uptake of [3H] thymidine and cell DNA synthesis during the early multiplication phase of herpesvirus hominis in BHK cells.

Experiments about the interaction of herpes viruses with BHK-cells during the first 6 h after infection concerning uptake and incorporation of dThd have been reported. During adsorption and penetration, the inhibition of uptake and of incorporation of [3H] dThd is sensitive to heat, but not to ultraviolet irradiation or cycloheximide. The eclipse is characterized by a strongly increased uptake of [3H] dThd and by inhibition of cell DNA synthesis. Both are sensitive to ultraviolet irradiation of the particles and cycloheximid treatment of the cells. It is concluded that the events during adsorption and penetration are dependent on the particles themselves, whereas the events during the eclipse depend on the activity of the viral genome. The implications of the findings are discussed.

Influence of dibutyryl cyclic AMP on thymidine uptake by herpes simplex virus infected cells and the intracellular level of cyclic AMP.

Dibutyryl cyclic AMP inhibits the increase of dThd and BrdUrd transport normally observed after infection with Herpesvirus hominis, type I and II. Incorporation is also reduced. Inhibition of uptake is non-competitive as analysed by the Lineweaver-Burk plot. Addition of this drug to infected cells also reduces the activity of the thymidine kinase (EC 2.7.1.75). Transport of dUrd, dCyd and dAdo is not reduced. 4--8 h after infection with thymidine kinase (+) herpes strains the level of cAMP increases. On infection with a thymidine kinase (-) virus, only a small elevation of cAMP can be shown. It was also found that early addition of actinomycin D or of cycloheximide prevents the increase of the cAMP level. This increase seems to depend on the activity of the herpes genome, because ultraviolet irradiation of infective particles destroys this ability.

The IL-12 response to herpes simplex virus is mainly a paracrine response of reactive inflammatory cells.

Herpes simplex virus (HSV) infection results in rapid and sustained up-regulation of interleukin (IL)-12, but the primary cellular source of IL-12 after HSV infection is unknown. We demonstrate that this cytokine largely derives from inflammatory cells rather than from productively infected epithelial cells. For optimal IL-12 induction, epithelial cells needed to be infected with replication-competent virus, and cells needed to be able to synthesize proteins. Our results also indicate that HSV-infected cells generate intermediary products that signal recruited inflammatory cells, which themselves were not HSV-infected, to generate IL-12. Possible mechanisms by which infected cells communicate with inflammatory cells to cause IL-12 production are discussed.

Activation of the radiosensitive EGR-1 promoter induces expression of the herpes simplex virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir.

Herein we describe experiments showing that the early growth response gene 1 (EGR-1) promoter is sufficient to confer selective expression of the luciferase gene (Luc) in glioma cell lines exposed to ionizing radiation. Activity of the EGR-1 promoter was investigated in human glioblastoma cells using the plasmid vector, pEGR-Luc. The EGR-1 promoter gene directed radiosensitive expression of luciferase. This promoter showed high levels of activity (10-fold) in irradiated glioma cell lines as compared to basal levels of activity in nonirradiated cell lines. Maximum activation was detectable at 1-3 hr after stimulation with 20 Gy. The results also demonstrate that cells modified to contain the herpes simplex virus-thymidine kinase (HSV-tk) gene under control of the EGR-1 promoter become sensitive to treatment with the antiviral agent ganciclovir (GCV), whereas nonirradiated cells and nontransfected cells were unaffected by this agent. This results suggest that therapeutic genes can be expressed selectively in irradiated glioma cells. The results also indicate that the EGR-1 promoter can be used to induce exogenous genes selectively in radiation fields used for the treatment of malignant brain tumors.

Virus replication and induction of interferon in human epidermal keratinocytes following infection with herpes simplex virus.

Keratinocyte cultures derived from surgical skin specimens of healthy newborns and adults were infected with herpes simplex virus (HSV) type 1 or 2. Typical HSV cytopathic effects involved all cell layers in stratified colonies, and paralleled the production of infectious virus. Virus growth curves and production of virus were comparable in newborn and adult keratinocytes. Interferon (IF) production by keratinocytes paralleled the yield of virus over at least 72 h, and was greater in cultures of adult cells than cultures from newborns. UV irradiation of HSV resulted in progressive virus inactivation and a parallel reduction in induced IF. This suggests that IF production was related to virus replication, and that irradiated (noninfectious) HSV DNA did not contribute significantly to the generation of IF in this system. These results establish that human epidermal keratinocytes can serve as a model system for quantitative assessment of herpes simplex virus infection.

Herpes simplex virus induces appearance of interferon-alpha/beta-producing cells and partially interferon-alpha/beta-dependent accumulation of leukocytes in murine regional lymph nodes.

As in vivo experimental system involving the local induction of interferon-alpha/beta (IFN-alpha/beta) responses was established in mice by injecting s.c. ultraviolet (UV)-inactivated herpes simplex virus (HSV) in the right ears, the left ears receiving phosphate-buffered saline (PBS) as a control. Circulating IFN-alpha/beta was present in blood as early as 6 h postinjection, and little or none was found 24 h postinjection. Identification of IFN-alpha/beta-producing cells, carried out by immunohistochemistry and in situ hybridization, demonstrated that the IFN response occurred mainly in the lymph node draining the HSV-injected ear and not in the contralateral lymph node. Occasionally, IFN-alpha/beta-producing cells were found in the spleen and in the skin. The injected HSV caused an inflammatory reaction in the skin and an almost threefold enlargement of the draining lymph node within 6 h. The latter was characterized by a general accumulation of all major lymphocyte subsets and a striking infiltration of neutrophils. Injection s.c. of neutralizing anti-IFN-alpha/beta antibodies before HSV injection reduced the increase in size of the draining lymph node by approximately 50% at 6 h, and no significant effects were seen at 24 h. The localization of cells producing IFN-alpha/beta in the lymph node and the capacity of such IFN-alpha/beta to at least partially mediate an early accumulation of cells suggest that the local IFN-alpha/beta response may have an important role in the initiation of early antiviral immune responses.

Lysis of herpes simplex virus (HSV) infected targets. III. Effect of HSV on natural killer activity.

Freshly isolated peripheral blood lymphocytes from patients (PBL-P) with recurrent herpetic corneal and skin lesions, and controls (PBL-C) with no recollection of herpetic disease, effectively lysed herpes simplex virus type 1 (HSV-1) infected but not uninfected allogeneic fibroblasts. However, after 48 hr of stimulation with HSV-1, the lytic activity of PBL-C declined, while that of PBL-P increased. PBL-P and PBL-C produced similar amounts of interferon when stimulated with HSV-1. Thus, the decline in lytic activity by HSV-1 stimulated PBL-C was not due to a lack of interferon production. Interestingly, the lytic activity of HSV-1 stimulated PBL appeared to be directed against a component commonly expressed on both uninfected and HSV-1 infected fibroblasts. This was indicated by the fact that after incubation with HSV-1, PBL-P lysed the uninfected fibroblasts as effectively as the HSV-1 infected fibroblasts. The augmented lysis of uninfected fibroblasts was not due to infection of the "uninfected fibroblasts" by HSV-1 carried over or produced by the HSV-1 stimulated PBL. Furthermore, the capacity of HSV-1 to augment lytic activity was not diminished markedly by ultraviolet irradiation, suggesting that augmentation is due to the stimulatory capacity rather than the infectivity of the virus. Our data show that interaction of PBL-P with HSV-1 results in enhancement of nonspecific lytic activity and loss of the capacity to discriminate HSV-1 infected from uninfected allogenic fibroblasts.

Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultraviolet B radiation.

The authors characterized a murine model of herpes simplex virus (HSV) reactivation in which recurrent herpetic keratitis was obtained in up to 80% of animals. Five weeks after ganglionic latency was established in National Institutes of Health inbred mice after corneal inoculation, HSV type 1 (HSV-1) was reactivated by irradiating the previously inoculated eye with ultraviolet (UV) light. Comparison of different UV wavelengths showed UVB to be optimal for reactivation, with peak viral recurrence being induced by a total exposure of approximately 250 mJ/cm2. Reactivated infectious virus generally began to appear in trigeminal ganglia 2 days postirradiation and was subsequently detectable in the cornea by both corneal swabbing and immunostaining for viral antigens. Two consecutive outbreaks of viral recurrence at the ocular surface were induced in selected animals by serial exposure to UVB. Advantages of this model over other models of recurrent keratitis are discussed.

Corneal Langerhans cell dynamics after herpes simplex virus reactivation.

PURPOSE: The authors investigated the progressive changes in the distribution of corneal Langerhans cells (LC) after reactivation of latent herpes simplex virus type 1 (HSV-1) in mice. METHODS: After corneal inoculation of National Institutes of Health inbred mice with HSV-1 and the establishment of latency, viral reactivation was induced by irradiating the ocular surface with 250 mJ/cm2 of ultraviolet B (UV-B) light. RESULTS: Subsequent viral replication in the cornea was followed by the migration of the LC toward the paracentral and central corneal epithelium. These areas are normally devoid of LC. The number of LC in the paracentral and central regions of the eye reached a peak at day 14 post-UV-B irradiation. After UV-B irradiation of mice latently infected with HSV-1, the development of corneal stromal opacification and neovascularization closely followed the migration of LC toward the central cornea and paralleled the influx of T-cells into the corneal stroma. This pattern was not observed in irradiated uninfected mice. CONCLUSIONS: LC migrate centrally in the corneal epithelium after viral reactivation. There is a direct correlation between the number of LC in the cornea and the degree of persistent stromal opacification.

Functions and proteins of herpes simplex virus type-1 that are involved in raising the mutation frequency of infected cells.

When cells are infected by herpes simplex virus type-1 (HSV-1) the mutation frequency is increased. To find which functions of the virus are responsible for this, a variety of viral strains and one fragment of viral DNA were tested in a mutagenesis assay. Mutagenesis was dependent on the binding of the virus to the cell surface and disassembly of the virus particle, but expression of virus genes was not necessary. Since this implied that mutagenesis was a result of the exposure of the interior of the cell to an internal structural component of the virus, the role of two likely components was examined. The host-shutoff function of the virus was not required for mutagenesis. However, a fragment of DNA from within the minimum transforming region of HSV-1 that encodes a possible virion protein was mutagenic when expressed from a eukaryotic expression vector. The encoded product of this DNA fragment is therefore a candidate for a transforming protein of HSV-1, and is the only protein currently suggested to be responsible for that function.