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.

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.

Prevention of viral myocarditis with recombinant human leukocyte interferon alpha A/D in a murine model.

Effects of recombinant human leukocyte interferon alpha A/D on experimental myocarditis due to encephalomyocarditis virus were investigated. Plaque reduction assays revealed that 50% of plaque formation in vitro in human amnion (FL) cells was inhibited by interferon alpha A/D (9.7 U/ml) when it was administered 24 hours before infection with the encephalomyocarditis virus. Four week old male DBA/2 mice were inoculated intraperitoneally with 10 plaque-forming units (pfu) of encephalomyocarditis virus. Interferon alpha A/D was administered subcutaneously (10(2) U/g body weight per day in Group 1, 10(3) U/g per day in Group 2 and 10(4) U/g per day in Group 3) starting 1 day before infection. It was also administered starting the same day in Group 4 and 1 day after virus inoculation in Group 5 (10(4) U/g per day in both groups). Control mice were injected with saline solution. Each group consisted of 10 mice; they were killed on day 4 for evaluation. Myocardial virus titers were significantly lower in Group 3 (8.2 +/- 25.2 X 10(2) pfu/mg, p less than 0.05) and Group 4 (3.0 +/- 5.5 X 10(3) pfu/mg, p less than 0.05) than in control mice (5.6 +/- 4.1 X 10(4) pfu/mg). Histologic examination showed extensive myocardial necrosis and cellular infiltration in all control mice, but no myocardial necrosis or cellular infiltration in Group 3 and less severe necrosis and infiltration in Group 4. There were no significant differences in myocardial virus titers or histologic changes between control mice and Group 1, 2 or 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Ribavirin treatment of murine coxsackievirus B3 myocarditis with analyses of lymphocyte subsets.

The efficacy of the synthetic nucleoside ribavirin in the therapy of experimental myocarditis caused by coxsackievirus B3 and its effects on peripheral and splenic lymphocyte subsets were investigated. Two week old male C3H/He mice were inoculated with coxsackievirus B3. Ribavirin was administered subcutaneously on days 0 to 15 (experiment I) and on days 4 to 15 (experiment II) in a dose of 200 (experiment I: Group 2, n = 10; experiment II: Group 5, n = 10) or 400 mg/kg per day (experiment I: Group 3, n = 10; experiment II: Group 6, n = 10). Control mice (experiment I: Group 1, n = 15; experiment II: Group 4, n = 14) received an injection of saline solution. In experiment I, mice treated with ribavirin survived significantly longer than did control mice (p = 0.005, Group 1 versus Group 2; p = 0.001, Group 1 versus Group 3). In experiment II, the survival rate on day 15 in ribavirin-treated mice was high compared with that in control mice (Group 4 = 14.3%, Group 5 = 20%, Group 6 = 50%). Myocardial viral titers on days 5 to 8 were significantly lower in ribavirin-treated mice of both experiments than in control mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of lymphocytes cultured from murine viral myocarditis specimens: a preliminary and technical report.

Long-term culture of lymphocytes from myocardial biopsy specimens has been reported. To test the usefulness of this technique in experimental models of murine myocarditis, the culture and characterization of lymphocytes from mice infected with encephalomyocarditis virus or coxsackievirus B3 were studied. Lymphocytes were successfully cultured from three hearts of encephalomyocarditis virus-infected mice in interleukin 2-containing culture. After approximately 10 days, lymphocytes migrated out of myocardium in the chronic stage of myocarditis and gradually increased in numbers. More than half of the exuding cells were Thy 1.2-positive. None of the myocardial samples from coxsackievirus B3-infected mice grew out lymphocytes. Pathologic examination of all specimens showed myocardial necrosis with lymphoid infiltration. Although further studies are needed, this preliminary study suggests that the technique of lymphocyte culture may promote new insights into the pathogenesis of experimental myocarditis.

Prevention of murine coxsackie B3 viral myocarditis and associated lymphoid organ atrophy with recombinant human leucocyte interferon alpha A/D.

The effects of recombinant human leucocyte interferon alpha A/D on experimental myocarditis due to coxsackie virus B3 were investigated. Specific plaque reduction assays showed that 50% of in vitro plaque formation in VERO (kidney of African green monkey) cells was inhibited by interferon alpha A/D 48 U.ml-1 when administered 24 h before infection with coxsackie virus B3. Three week old male C3H/He mice were inoculated intraperitoneally with 3 x 10(2) plaque-forming units (pfu) of coxsackie virus B3. Controls (group 1) were injected with saline. Interferon alpha A/D 10(4) (group 2) or 10(3) (group 3) U.g-1.day-1, was administered subcutaneously daily, starting 1 d before the infection. Interferon alpha A/D 10(4) U.g-1.day-1 (group 4) was also given, starting the same day. Each group consisted of ten mice. Animals were sacrificed on d 5 for evaluation. Myocardial virus titres were significantly lower in groups 2, 3, and 4 (p less than 0.05) compared with controls. Histological examination showed extensive myocardial necrosis and cellular infiltration in all untreated mice but less severe necrosis and cellular infiltration in treated groups. Thymus and spleen weights in treated groups were greater than in the untreated group, and cellular depletion was less. Thus interferon alpha A/D effectively inhibited myocardial replication of coxsackie virus B3, reduced the myocardial inflammatory response, and prevented the disease associated lymphoid organ atrophy in this animal model.

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.

Cytokine dysregulation in AIDS: in vivo overexpression of mRNA of tumor necrosis factor-alpha and its correlation with that of the inflammatory cytokine GRO.

The human immunodeficiency virus establishes an intimate interaction with the immune system. The virus can use cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (Il-1), to regulate its own expression by modifying the normal immunoregulatory network. We demonstrate that mRNA of the cytokine TNF-alpha from peripheral blood mononuclear cells is overexpressed in virtually all patients with AIDS who do not have active opportunistic infections compared with uninfected volunteers (p < 0.0001). This overexpression correlates with elevated mRNA levels of the recently discovered GRO (p < 0.05), a cytokine involved in the inflammatory response.

Progressive polyradiculopathy in acquired immune deficiency syndrome.

We studied three patients with acquired immune deficiency syndrome (AIDS) and progressive polyradiculopathy. Postmortem examination of one patient disclosed extensive necrosis, inflammatory infiltrates, and focal vasculitis of spinal roots. Typical cytomegaloviral (CMV), intranuclear, and intracytoplasmic inclusions were noted within enlarged endoneurial and endothelial cells. Progressive polyradiculopathy is an unusual complication of AIDS; CMV may be the causative agent in certain cases.

Mechanism of action of foscarnet against viral polymerases.

Foscarnet is a pyrophosphate analogue with activity against herpesviruses, human immunodeficiency virus (HIV), and other RNA and DNA viruses. Foscarnet and its analogues achieve their antiviral effects via inhibition of viral polymerases, with such inhibition not being dependent on activation or phosphorylation of the compounds by viral or cellular proteins. Current evidence indicates that foscarnet interferes with exchange of pyrophosphate from deoxynucleoside triphosphate during viral replication by binding to a site on the herpesvirus DNA polymerase or HIV reverse transcriptase. Reviewed herein are basic findings regarding the mechanism of action and antiviral activity of foscarnet and the related compound phosphonoacetic acid (PAA), as well as findings regarding potential mechanisms of viral resistance and interactions with other antiviral agents.

Topical 5 percent acyclovir in polyethylene glycol for herpes simplex labialis. Antiviral effect without clinical benefit.

A double-blind, placebo-controlled trial of topical 5 percent acyclovir in polyethylene glycol was conducted among 208 patients with herpes simplex labialis. Reduced excretion of virus from lesions was seen in the subgroup of patients who entered the study within eight hours of lesion onset, but no differences were noted in the patients who began treatment nine to 25 hours after onset. No clinical benefit from treatment with acyclovir was observed.

Drug-resistant herpes simplex virus in vitro and after acyclovir treatment in an immunocompromised patient.

An acyclovir-resistant herpes simplex virus (HSV) has been isolated from an immunocompromised patient during treatment for severe orofacial HSV infections with acyclovir. The resistant virus is deficient in expression of the HSV thymidine kinase (TK). Emergence of acyclovir resistance during clinical use appears to parallel the in vitro observations of selection for TK-deficient, acyclovir-resistant viruses following serial passage in the presence of the drug. The clinical importance of drug-resistant HSV in unclear, and further investigations are required to determine whether it will be an impediment to successful therapy of HSV infections.

Enhanced survival of ultraviolet-irradiated herpes simplex virus in cells exposed to antiviral agents.

Enhanced survival of UV-irradiated HSV-1 is demonstrated in monkey cells exposed to inhibitors of viral DNA synthesis. Phosphonoacetic acid (PAA), adenine arabinoside (ara-A), and cytosine arabinoside (ara-C) pretreatment of infected cells is associated with concentration-dependent reactivation of UV-HSV-1. At concentrations that result in enhanced virus survival, inhibition of cell DNA synthesis is observed by either ara-A or ara-C, but not by PAA. Pretreatment of uninfected cells with acycloguanosine (ACG) is not associated with reactivation of irradiated HSV-1, and this is probably due to insufficient generation of ACG-triphosphate, the active inhibitor of viral and cell DNA synthesis.

Significance of resistance of herpes simplex virus to acyclovir.

The genome of herpes simplex virus codes for several enzymes, including viral thymidine kinase and viral deoxyribonucleic acid (DNA) polymerase. When viral resistance develops, it does so by changes in these two enzymes. Three possible mechanisms of viral resistance to acyclovir include (1) selection of viral mutants that make little or no thymidine kinase and do not phosphorylate acyclovir adequately, (2) selection of mutants that can phosphorylate thymidine but cannot phosphorylate acyclovir (i.e., these viruses have thymidine kinases with altered substrate specificity), and (3) selection of viruses that have altered DNA polymerases that replicate viral DNA in the presence of acyclovir triphosphate. Thymidine kinase-deficient virus has been isolated from clinical isolates frequently, but few strains appear to be virulent for animals or humans and only a few seem to have caused clinical disease. Viruses with altered substrate specificity have been reported but viruses with an altered DNA polymerase have not occurred in clinical practice. Antiviral drugs should be used only when necessary to minimize the appearance of resistant strains of virus.

Analysis of the thymidine kinase gene from clinically isolated acyclovir-resistant herpes simplex viruses.

The isolation and description of acyclovir-resistant (ACVR) herpes simplex-2 viruses from patients with AIDS has recently been reported. These ACVR viruses were all markedly decreased in their thymidine kinase (TK) activity, and 6 of 10 of these TK viruses were able to establish latency. In addition, one of these isolates, ACVR-86012 was neuropathogenic in a murine encephalitis model. In this paper, the characteristics of these isolates with respect to TK polypeptide synthesis are examined. All but one isolate synthesized a detectable TK protein by immunoprecipitation, and 9/10 of the TK proteins had an altered electrophoretic mobility as compared to wild-type. The TK polypeptide from the neuropathogenic isolate ACVR-86012 was full-length and the gene was sequenced. An amino acid change from a glutamine to a proline at amino acid residue 105 was detected compared to the wild-type HSV-333 strain. These results indicate that an amino acid change in the NH2 portion of the TK protein is associated with a full-length peptide with decreased enzyme activity but the virus retains neuropathic virulence.