Antiherpesvirus activities of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine (A-5021) in cell culture

Antiherpetic activity of (1'S,2'R)-9-([1',2'-bis(hydroxymethyl)cycloprop-1'yl]methyl)guanine (A-5021) was compared with those of acyclovir (ACV) and penciclovir (PCV) in cell cultures. In a plaque reduction assay using a selection of human cells, A-5021 showed the most potent activity in all cells. Against clinical isolates of herpes simplex virus type 1 (HSV-1, n = 5) and type 2 (HSV-2, n = 6), mean 50% inhibitory concentrations (IC50s) for A-5021 were 0.013 and 0.15 microgram/ml, respectively, in MRC-5 cells. Corresponding IC50s for ACV were 0.22 and 0.30 microgram/ml, and those for PCV were 0.84 and 1.5 micrograms/ml, respectively. Against clinical isolates of varicella-zoster virus (VZV, n = 5), mean IC50s for A-5021, ACV, and PCV were 0.77, 5.2, and 14 micrograms/ml, respectively, in human embryonic lung (HEL) cells. A-5021 showed considerably more prolonged antiviral activity than ACV when infected cells were treated for a short time. The selectivity index, the ratio of 50% cytotoxic concentration to IC50, of A-5021 was superior to those of ACV and PCV for HSV-1 and almost comparable for HSV-2 and VZV. In a growth inhibition assay of murine granulocyte-macrophage progenitor cells, A-5021 showed the least inhibitory effect of the three compounds. These results show that A-5021 is a potent and selective antiviral agent against HSV-1, HSV-2, and VZV.     

Mode of action of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl) cycloprop-1'-yl]methyl]guanine (A-5021) against herpes simplex virus type 1 and type 2 and varicella-zoster virus

The mode of action of (1'S,2'R)-9-([1', 2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl)guanine (A-5021) against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) was studied. A-5021 was monophosphorylated at the 2' site by viral thymidine kinases (TKs). The 50% inhibitory values for thymidine phosphorylation of A-5021 by HSV-1 TK and HSV-2 TK were comparable to those for penciclovir (PCV) and lower than those for acyclovir (ACV). Of these three agents, A-5021 inhibited VZV TK most efficiently. A-5021 was phosphorylated to a mono-, di-, and triphosphate in MRC-5 cells infected with HSV-1, HSV-2, and VZV. A-5021 triphosphate accumulated more than ACV triphosphate but less than PCV triphosphate in MRC-5 cells infected with HSV-1 or VZV, whereas HSV-2-infected MRC-5 cells had comparable levels of A-5021 and ACV triphosphates. The intracellular half-life of A-5021 triphosphate was considerably longer than that of ACV triphosphate and shorter than that of PCV triphosphate. A-5021 triphosphate competitively inhibited HSV DNA polymerases with respect to dGTP. Inhibition was strongest with ACV triphosphate, followed by A-5021 triphosphate and then (R,S)-PCV triphosphate. A DNA chain elongation experiment revealed that A-5021 triphosphate was incorporated into DNA instead of dGTP and terminated elongation, although limited chain extension was observed. Thus, the strong antiviral activity of A-5021 appears to depend on a more rapid and stable accumulation of its triphosphate in infected cells than that of ACV and on stronger inhibition of viral DNA polymerase by its triphosphate than that of PCV.     

Prevalence of herpes simplex virus type 1- and 2- specific antibodies among the acute, recurrent, and provoked types of female genital herpes

Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P < 0.005), and 55% for the recurrent type (P < 0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.     

DNA immunization against experimental genital herpes simplex virus infection

A nucleic acid vaccine, expressing the gene encoding herpes simplex virus (HSV) type 2 glycoprotein D (gD2) under control of the cytomegalovirus immediate-early gene promoter, was used to immunize guinea pigs against genital HSV-2 infection. The vaccine elicited humoral immune responses comparable to those seen after HSV-2 infection. Immunized animals exhibited protection from primary genital HSV-2 disease with little or no development of vesicular skin lesions and significantly reduced HSV-2 replication in the genital tract. After recovery from primary infection, immunized guinea pigs experienced significantly fewer recurrences and had significantly less HSV-2 genomic DNA detected in the sacral dorsal root ganglia compared with control animals. Thus, immunization reduced the burden of latent infection resulting from intravaginal HSV-2 challenge, and a nucleic acid vaccine expressing the HSV-2 gD2 antigen protected guinea pigs against genital herpes, limiting primary infection and reducing the magnitude of latent infection and the frequency of recurrent disease.     

Rotational instability and integrity of the interosseous membrane in cadaveric ulnar shaft fractures

We demonstrate that the novel immunosuppressive agent mycophenolate mofetil (MMF), that has been approved for use in kidney transplant recipients, strongly potentiates the antiviral activity of acyclovir in murine models for herpesvirus infections. Hairless mice that were infected intracutaneously with herpes simplex virus type 1 were treated systemically with ACV (20 mg/kg per day) and topically with 5% MMF. Combined use of both drugs resulted in an almost complete protection, whereas single use of either compound had virtually no effect. When athymic-nude mice were infected with an ACV-resistant (ACVr)-thymidine kinase-deficient (TK-) HSV-2 strain, combined use of systemically administered ACV (100 mg/kg per day) and topically applied MMF (5%) protected 60% of the animals against the infection, whereas all mice treated with either drug alone succumbed. Since transplant recipients under MMF therapy may develop opportunistic herpesvirus infections, requiring treatment with acyclovir (or valaciclovir), our findings have important implications for the treatment of these herpesvirus infections.     

The prophylactic effect of immunization with DNA encoding herpes simplex virus glycoproteins on HSV-induced disease in guinea pigs

Plasmid expression vectors encoding herpes simplex virus type 2 (HSV-2) glycoproteins B (gB) or D (gD) were constructed and tested for their ability to immunize guinea pigs against genital HSV infection. Immunization with a plasmid expressing the aminoterminal 707 amino acids (aa) of gB induced humoral immune responses detected by ELISA and virus neutralization. When challenged by vaginal infection, immunized animals were partially protected from genital herpes, exhibiting significantly reduced primary and subsequent recurrent disease. When the gB plasmid was combined with a plasmid expressing full-length gD, immunized guinea pigs developed humoral responses to both proteins and were also significantly protected from viral challenge.     

The effect of gramicidin, a topical contraceptive and antimicrobial agent with anti-HIV activity, against herpes simplex viruses type 1 and 2 in vitro

The effect of an anti-HIV compound, gramicidin, previously used as a topical antibiotic and vaginal contraceptive, on the replication of herpes simplex viruses (HSV) type 1 and 2 has been examined. Human WI-38 fibroblasts were inoculated with either HSV type in the presence of serial dilutions of gramicidin and reduction in viral yield was measured by ELISA. The 50% inhibitory dose (IC50) of gramicidin against 3 HSV-1 and 4 HSV-2 isolates was equal to 0.3 microgram/ml and was comparable to the efficacy of the anti-HSV agent acyclovir (ACV). The IC50 of gramicidin required to protect WI-38 from cytolytic effect of HSV was 10 micrograms/ml at day 5 postinfection, indicating that at this time point the activity of gramicidin was inferior than that of ACV. Nevertheless, gramicidin suppressed the replication of ACV-resistant thymidine kinase and DNA polymerase HSV mutants at doses effective against ACV-sensitive strains. The results suggest that the antimicrobial and spermostatic agent, gramicidin, has potential against sexually transmitted diseases (STDs) and for prophylaxis of sex-borne HIV and HSV infections.     

Differential mechanism of cytostatic effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, and other antiherpetic drugs on tumor cells transfected by the thymidine kinase gene of herpes simplex virus type 1 or type 2

After they have been transfected with the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) thymidine kinase (TK) gene murine mammary carcinoma (FM3A) cells become highly sensitive to the growth inhibitory properties of the antiherpetic agents (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 9(-)[(2-hydroxyethoxy)methyl]guanine (acyclovir, ACV), 9(-)[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, ganciclovir), and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU). BVDU was 100-fold more potent an inhibitor of HSV TK gene-transfected tumor cell growth (50% inhibitory concentration (IC50), 0.0020-0.0047 microM) than FMAU or DHPG (IC50, 0.051-0.277 microM) and 1000-fold more potent than ACV (IC50, 0.42-4.9 microM). As a rule, the test compounds were more cytostatic to HSV-2 TK than HSV-1 TK gene-transfected FM3A cells. This may be ascribed to the higher phosphorylating capacity (Vmax/Km) of HSV-2 TK than HSV-1 TK and/or to the higher TK enzyme levels of the HSV-2 TK gene-transfected FM3A cells than the HSV-1 TK gene-transfected FM3A cells. Thymidylate synthase of the HSV TK gene-transfected FM3A cells appears to be the target enzyme for the cytostatic action of BVDU, but not FMAU, DHPG, or ACV. Instead, the cytostatic activity of DHPG seems to be correlated with its conversion to the triphosphate form and subsequent incorporation into the DNA of HSV TK gene-transfected FM3A cells.     

Distribution of herpes simplex virus type 1 and 2 genomes in the human spinal ganglia

Herpes simplex virus (HSV) is well known for its propensity to cause recurrent oral or genital mucosal infections in humans. HSV-1 is involved primarily in oral lesions, whereas HSV-2 is more frequently involved in genital lesions. Based on this, it is thought that HSV-1 may produce latent infections in trigeminal ganglia, and HSV-2 in the sacral ganglia. However the distribution pattern of latent HSV-1 and HSV-2 infections in spinal ganglia remains unknown. Using the polymerase chain reaction we detected latent herpes HSV-1 and HSV-2 in human spinal ganglia obtained from autopsy material. A pair of primers which were specific for a part of the HSV-1 and HSV-2 DNA polymerase domain were employed. HSV-1 and HSV-2 DNAs were detected in 11 of 40 (28%) and 15 of 40 (38%) cervical ganglia, respectively, 52 of 103 (50%) and 47 of 103 (46%) thoracic ganglia, 16 of 53 (30%) and 17 of 53 (32%) lumbar ganglia, and 3 of 20 (15%) and 3 of 20 (15%) sacral ganglia. These findings suggest that latent HSV-1 and HSV-2 infections have a widespread distribution from the cervical ganglia to sacral ganglia. Importantly this study demonstrated latent HSV-1 infection of both the lumbar and sacral ganglia for the first time.     

Herpes simplex virus type 2 infections presenting as brainstem encephalitis and recurrent myelitis

We describe here 3 patients with central nervous system infection caused by herpes simplex virus type 2 (HSV-2); one patient with brainstem encephalitis and two with recurrent transverse thoracic myelitis. All three patients showed increased IgG antibodies to HSV in the cerebrospinal fluid (CSF). HSV-2 DNA was demonstrated in the CSF by polymerase chain reaction (PCR) amplification. Upon treatment with acyclovir, one patient with myelitis partially recovered and the others completely recovered. It is important to recognize the wide spectrum of clinical manifestations of HSV-2 infection in the central nervous system (CNS).     

Rapid screening tests for determining in vitro susceptibility of herpes simplex virus clinical isolates

The susceptibility of human herpes simplex virus (HSV) to acyclovir (ACV) was determined with the use of a single dose of the drug (1 and 2 micrograms of ACV per ml for HSV-1 and HSV-2, respectively) in two rapid assays: a rapid cytopathic effect inhibitory assay (Rapid CIA) and a rapid dye uptake assay (Rapid DUA). These tests allow the simultaneous determination of virus titer and susceptibility to ACV at a determined viral concentration (100 50% tissue culture infective doses and 100 50% dye uptake units). These tests were compared with a conventional susceptibility assay (dye uptake assay) and showed similar results. Indeterminate results with the Rapid CIA appeared in 3 of 30 samples. With the use of both Rapid CIA and Rapid DUA, we were able to determine the susceptibility of 100% of the isolates. The rapid tests, unlike conventional assays, are able to provide susceptibility results within 3 days after the virus has been isolated from a clinical specimen and could thus play a direct role in therapeutic decisions.     

Chemistry and anti-herpes simplex virus type 1 evaluation of cycloSal-nucleotides of acyclic nucleoside analogues

The synthesis of different cycloSal-phosphotriesters of the acyclic nucleoside analogues acyclovir (ACV), penciclovir (PCV) and T-penciclovir (T-PCV) as potential new lipophilic, membrane-soluble pronucleotides is described. The introduction of the cycloSal moiety was achieved by using reactive cyclic chlorophosphane reagents. In addition to the cycloSal-PCV monophosphate (MP) phosphotriesters, a second derivative bearing an acetyl group at the second primary alcohol function was prepared. In hydrolysis studies the cycloSal-ACVMPs showed the expected range of hydrolytic stability dependent on the substituent in the masking group (8-17 h). In contrast, the cycloSal-PCVMP derivatives exhibited a 11- to 15-fold increase in hydrolytic lability as compared to the corresponding cycloSal-ACVMP derivatives. We demonstrated that the free primary alcohol group is responsible for this rate acceleration because cycloSal-OAc-PCVMP, in which the hydroxyl group was blocked by acetylation, did not show the aforementioned acceleration. Unexpectedly, the hydrolysis product was not PCVMP but according to NMR and mass spectrometry it was cycloPCVMP (cPCVMP). The title compounds were evaluated in vitro for their ability to inhibit herpes simplex virus type 1 (HSV-1) and thymidine kinase-negative (TK-) HSV-1 replication in Vero cells. The cycloSal-ACVMP compounds exhibited high antiviral activity in HSV-1-infected cells. More importantly, one derivative retained all activity from the wild-type virus strain in HSV-1/TK(-)-infected Vero cells. The PCV derivatives were markedly less active. The reason for the failure of the cycloSal-PCVMPs seems to be due to the formation of cPCVMP instead of the desired PCVMP.     

Synthesis of adenine derivatives and their activities against herpes virus in vitro

A series of 9-(N4-substituted acetaldehyde thiosemicarbazone) adenines were synthesized and evaluated for antiherpes virus activity. Compounds 4a-l were prepared by condensation of 9-(acetaldehyde) adenine(6) and the corresponding N4-substituted thiosemicarbazides (10). The antiviral effects of all compounds 4a-l were tested in vitro in primary rabbit kidney cell cultures infected with herpes simplex virus type 1 (HSV-1) and varicella-herpes zoster virus (VZV), and in primary human embryo cell cultures infected with herpes simplex virus type 2 (HSV-2). The results showed that the minimum inhibitory concentrations (MIC) of 4e and 4f for HSV-1 and VZV were 20, 40, 20 and 20 micrograms.ml-1, respectively, and other compounds were 200 micrograms.ml-1. For HSV-2, the MIC of all tested compounds were 300 micrograms.ml-1. We also evaluated the antiherpetic effect of 4e (and 4f) by combination with acyclovir (ACV) in the ratio of 1:1 in vitro. The MIC of the combined compounds were 2 micrograms.ml-1 for 4e and 6 micrograms.ml-1 for 4f, while their minimum cytotoxicities (MCC) in the cell were markedly reduced compared with the individual compounds.     

Development of antiviral therapeutic agents from traditional medicines

Traditional medicines contain various metabolites derived from nucleic acid, protein, and lipid metabolism. Some of these specific metabolites may recognize the differences between viral and host metabolism resulting in anti-viral activity; hence traditional medicines may be useful sources for new antiviral agents. Traditional medicines can be cheaply obtained and have been orally administered as hot-water extracts. Therefore, they may be used for the prophylactic and therapeutic treatment of viral infection by drinking them, such as coffee or tea. Here we describe how the antiviral activity of traditional medicines was screened in vitro and how their therapeutic antiviral activities were verified in vivo, to obtain traditional antiviral medicines that can be clinically used. Therefore, we have selected 12 herbal extracts, from more than 250 herbal medicines, that exhibit therapeutic activities against cutaneous herpes simplex virus (HSV) type 1 (HSV-1) infection in mice. Four of the 12 augmented the therapeutic efficacy of acyclovir (ACV) in mice and showed potent anti-HSV activity against infection with ACV-resistant HSV-1 mutants in mice. These herbal extracts selectively inhibited viral DNA synthesis and showed a different mode of anti-HSV-1 action from that of ACV. They were also effective against both recurrent HSV and cytomegalovirus infections, without toxicity. Such prophylactic and therapeutic antiviral activities of the traditional medicines were verified by the purification of major active compounds. We could show new indications of traditional medicines as antiviral agents. Thus, the drinking of the extracts, in a daily tea or coffee, may be used for prophylaxis and therapy of diseases caused by herpes virus infection and improve the quality of life.     

Opposite effects of tumour necrosis factor-alpha on type I and III collagen gene expression by human dermal fibroblasts in monolayer and three-dimensional cultures

An HIV-1 infected immunosuppressed patient (CD4+ cell counts: 382 cells/microL; viral load 94,000 copies/mL) with recurrent perianal herpes simplex virus type 2 (HSV-2) infections is described, showing an unusual exophytic tumour resembling a squamous cell carcinoma in the lateral part of the tongue. He also had persistent facial herpes infection, oral candidosis, oral hairy leukoplakia and lymphadenopathy. The presence of HSV-2 was detected by polymerase chain reaction both in smears and in a tissue biopsy taken from the involved tongue area. Treatment with brivudin, a new oral virustatic drug, led to rapid regression of the tumour.     

Detection of immunoglobulin M antibodies to glycoprotein G-2 by western blot (immunoblot) for diagnosis of initial herpes simplex virus type 2 genital infections

Western blots (immunoblots) for the detection of immunoglobulin M (IgM) antibodies specific for herpes simplex virus type 1 (HSV-1) and HSV-2 in patients' sera were developed. The locations of the type-specific glycoprotein G (gpG-2) of HSV-2 (92- and 140-kDa forms) and glycoprotein C of HSV-1 (gpC-1), which carries mostly type-specific antigenic epitopes, were checked with specific monoclonal antibodies. Western blot assays for IgM antibody to gpC-1 or gpG-2 were performed after depletion of IgG by precipitation with anti-human IgG. In patients with primary HSV-2 genital infections, seroconversion of IgM and IgG antibodies to both the 92- and 140-kDa forms of gpG-2 was observed, although both antibodies appeared in convalescent-phase serum after the first week. IgM and IgG antibodies to low-molecular-size polypeptides (40 to 65 kDa) were the first antibodies observed in patients with primary infection, but these antibodies were cross-reactive with HSV-1 and HSV-2. However, in patients with recurrent HSV-2 infections, IgG antibodies to both forms of gpG-2 and the low-molecular-size polypeptides were found no matter how early after onset the patient was bled, and IgM to gpG-2 did not appear. In patients with nonprimary initial genital HSV-2 infections, IgG antibody to HSV-1 was demonstrated in the first serum specimen, and HSV-2-specific IgM was found in 39% of the serum specimens. Hence, the Western blot assay can be used to test for IgM antibody to gpG-2, allowing for the retrospective diagnosis of inital HSV-2 infections and its use as a supplementary test to the gpG-2 IgG enzyme-linked immunosorbent assays developed elsewhere. In contrast, IgM antibody to gpG-2 is not usually detected in patients with recurrent HSV-2 infections.     

Evidence of latency and reactivation of both herpes simplex virus (HSV)-1 and HSV-2 in the genital region

While superinfection with different herpes simplex virus (HSV) types has been demonstrated in animals, the ability of the two HSV types to colonize and reactivate in the same anatomic region in humans has not been well demonstrated. In 6 patients, both HSV-1 and HSV-2 was recovered from genital lesions. In 4 of them, who initially acquired genital HSV-1 infection, subsequent HSV-2 infection presented as a prolonged episode of genital lesions and a marked increase in the frequency of genital recurrences. While most of the subsequent clinical reactivations were HSV-2, in 2 patients the recurrence rate of genital HSV-1 increased after the acquisition of HSV-2. These data demonstrate the ability of a second HSV type to infect the same anatomic region and illustrate the difference in reactivation frequency of the two types in the same person. Typing of HSV isolates may be useful in persons with recent alteration in recurrence rates of genital HSV.     

Protective vaccination against primary and recurrent disease caused by herpes simplex virus (HSV) type 2 using a genetically disabled HSV-1

The vaccine potential of a mutant herpes simplex virus (HSV) type 1, with a deletion in the glycoprotein H (gH) gene, was evaluated. The virus requires a gH-expressing cell line for multi-cycle growth but can complete a single cycle of infection in noncomplementing cells. Such viruses, termed DISC (disabled infectious single cycle) viruses, should be safe, yet still able to stimulate humoral and cell-mediated responses against a broad range of virus antigens in vaccinated hosts. Prophylactic vaccination of guinea pigs with DISC HSV-1, by ear scarification or direct infection of the vaginal mucosa, afforded a high degree of protection against HSV-2-induced primary genital disease and reduced significantly the frequency of subsequent disease recurrence. There was also a trend toward reduced recurrence following therapeutic vaccination of animals already infected with HSV-2. DISC HSV vaccination, therefore, offers an effective route for control of HSV disease.     

Induction of apoptosis in HEp-2 cells by infection with herpes simplex virus type 2

Although herpes simplex virus type 1 (HSV-1) does not induce apoptosis in infected HEp-2 cells, herpes simplex virus type 2 (HSV-2) did induce apoptosis in a small but significant fraction of the same cells. Apoptosis was not observed in Vero or HeLa cells infected with HSV-2. In addition, HSV-2 infection in the presence of cycloheximide induced extensive apoptosis of HEp-2 or HeLa cells.     

Advances in the treatment of herpesvirus infection: the role of famciclovir

Shingles (herpes zoster) is the result of reactivation of varicella-zoster virus after years of latency. The acute phase is self-limiting but is often associated with moderate-to-severe pain; postherpetic neuralgia is the most frequent and debilitating complication of shingles, occurring in 3.4 per 1000 individuals per year. In the case of genital herpes, herpes simplex virus can reactivate to cause recurrent episodes as often as several times a year, sometimes for the remainder of a person's life. Antiviral agents such as famciclovir, valacyclovir, and acyclovir can be used to shorten the course and decrease the severity of these diseases and may suppress the virus itself, thereby preventing future outbreaks of genital herpes. This article presents a brief synopsis of the etiology of herpes zoster and genital herpes and reviews 12 key studies that demonstrate the efficacy of famciclovir in the management of these two conditions.