Acyclovir-resistant Herpes Simplex Virus Research Articles

Herpes Simplex Encephalitis: From Virus to Therapy

Herpes simplex virus (HSV) is the cause of herpes simplex encephalitis (HSE), a devastating human disease which occurs in 2-4 cases per million/year. HSE results either from a primary infection or virus reactivation, in accordance with the common pattern of HSV infection which is a chronic lifelong process. However its pathophysiology remains largely unknown and its poor prognosis is in contrast with the usually good tolerance of most clinical herpetic manifestations. HSE is due to HSV type 1 (HSV-1) in most cases but HSV type 2 (HSV-2) may be also implicated, especially in infants in the context of neonatal herpes. Polymerase chain reaction detection of HSV DNA in cerebrospinal fluid is the diagnosis of choice for HSE. Acyclovir, a nucleoside analogue which inhibits viral DNA polymerase activity, is the reference treatment of HSE while foscarnet constitutes an alternative therapy and the efficacy of cidofovir is currently uncertain in that context. The emergence of HSV resistance to acyclovir, a phenomenon which is mainly observed among immunocompromised patients, is a current concern although no case of HSE due to an acyclovir-resistant HSV strain has been reported to date. Nevertheless the identification and development of novel therapeutic strategies against HSV appears to be a non dispensable objective for future research in virology.

Efficacy of plant products against herpetic infections

ZusammenfassungÄtherische Öle aus unterschiedlichen Heilpflanzen sind hoch wirksam gegen einige Virusinfektionen, z. B. gegen Herpes-simplex-Virus vom Typ 1, den Erreger des Lippenherpes. Melissenöl, Teebaumöl und Pfefferminzöl sind in vitro deutlich antiherpetisch wirksam. Dies beruht vor allem auf einer direkten Interaktion des Öls mit Viruspartikeln. Die genannten ätherischen Öle sind auch gegen aciclovirresistente Herpesstämme wirksam. In klinischen Studien wurde für Teebaumöl eine infektionshemmende Eigenschaft nachgewiesen. Letztere führt zur schnelleren Abheilung des Lippenherpes. Darüber hinaus wirken ätherische Öle bei topischer Anwendung auch schmerzlindernd und entzündungshemmend. Eine 3- bis 4-malige topische Applikation verdünnter ätherischer Öle wird zur Therapie bei herpetischen Infektionen empfohlen. Einige Pflanzenprodukte, z. B. aus der Melisse, sind bereits im Handel zur Behandlung von herpetischen Infektionen erhältlich.

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities.

Acyclovir-resistant herpes simplex virus treated with intravenous foscarnet and topical cidofovir

Acyclovir-resistant herpes simplex virus treated with intravenous foscarnet and topical cidofovir

Antiviral activity of the Lippia graveolens (Mexican oregano) essential oil and its main compound carvacrol against human and animal viruses.

Mexican oregano (Lippia graveolens) is a plant found in Mexico and Central America that is traditionally used as a medicinal herb. In the present study, we investigated the antiviral activity of the essential oil of Mexican oregano and its major component, carvacrol, against different human and animal viruses. The MTT test (3–4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide) was conducted to determine the selectivity index (SI) of the essential oil, which was equal to 13.1, 7.4, 10.8, 9.7, and 7.2 for acyclovir-resistant herpes simplex virus type 1 (ACVR-HHV-1), acyclovir-sensitive HHV-1, human respiratory syncytial virus (HRSV), bovine herpesvirus type 2 (BoHV-2), and bovine viral diarrhoea virus (BVDV), respectively. The human rotavirus (RV) and BoHV-1 and 5 were not inhibited by the essential oil. Carvacrol alone exhibited high antiviral activity against RV with a SI of 33, but it was less efficient than the oil for the other viruses. Thus, Mexican oregano oil and its main component, carvacrol, are able to inhibit different human and animal viruses in vitro. Specifically, the antiviral effects of Mexican oregano oil on ACVR-HHV-1 and HRSV and of carvacrol on RV justify more detailed studies.

Treatment of Acyclovir-Resistant Herpes Simplex Virus with Continuous Infusion of High-Dose Acyclovir in Hematopoietic Cell Transplant Patients

Infection because of herpes simplex virus (HSV) that is resistant to acyclovir (ACV) poses treatment challenges in hematopoietic cell transplant (HCT) patients. We present a series of patients with ACV-resistant HSV following HCT who were successfully treated with continuous infusion high-dose ACV after failing standard treatment regimens for ACV-resistant HSV.

Acyclovir resistance in herpes simplex encephalitis

Herpes simplex virus type 1 is a common cause of severe sporadic encephalitis. Treatment with acyclovir is highly effective in this disease. We report the case of a 27-year-old, immunocompetent woman with acyclovir-resistant herpes simplex encephalitis. Although she had not been treated before, herpes simplex virus type 1 DNA from the cerebrospinal fluid showed a non-synonymous mutation in the thymidine kinase gene, which is likely to have caused resistance to acyclovir. Herpes simplex encephalitis resolved after treatment with foscarnet. To our knowledge, this is the first report of acyclovir-resistant herpes simplex virus encephalitis in an immunocompetent, previously therapy-naive adult.

Successful Treatment of Acyclovir-Resistant Herpes Simplex Virus With Intralesional Cidofovir

Acyclovir-resistant herpes simplex virus (HSV) has become increasingly common, particularly among patients with human immunodeficiency virus (HIV). We present a case of acyclovir-resistant HSV treated with intralesional cidofovir.

Treatment of Severe Acyclovir-Resistant Herpes Simplex Virus Infection with Continuous Infusion of High Dose Acyclovir Following Hematopoietic Cell Transplantation.

Abstract 2224Poster Board II-201Although acyclovir (ACV)-resistant herpes simplex virus (HSV) occurs in healthy patients, higher rates have been reported in 7-27% of immunocompromised patients, particularly in the setting of acyclovir prophylaxis. Infection due to ACV-resistant HSV (ACV-R HSV) is frequently severe and poses challenges to treatment, particularly following hematopoietic cell transplantation (HCT). Unfortunately, currently available alternative therapies including foscarnet, cidofovir, and ganciclovir are frequently limited by their toxicity profile. We present a series of five HCT patients from 1997-2008 with severe infections due to ACV-R HSV who were successfully treated with continuous infusion of high dose acyclovir. A Pubmed search of published literature from 1948-2009 confirmed this as the first report of ACV-R HSV in HCT patients treated with this regimen.The characteristics of the patients are summarized in the table. Patients ranged in age from 24 to 55 and had undergone an allogeneic HCT following a myeloablative preparative regimen. All grafts were composed of peripheral blood hematopoietic cells. Underlying diseases included acute myelogenous leukemia, myelodysplastic syndrome, and essential thrombocytosis. Four of the five patients were receiving treatment for GVHD of the skin (grades 2-4), with regimens that included varying doses of prednisone. Infection was due to HSV-1 in three patients and HSV-2 in two. In vitro testing revealed that high median inhibitory concentrations were necessary to inhibit viral replication by 50% (MIC50) for all five isolates consistent with resistance to acyclovir, ganciclovir, and foscarnet. Failed HSV treatment regimens included standard, intermittent doses of acyclovir (5-10 mg/kg IV every eight hours), foscarnet, cidofovir, and/or famciclovir. Adverse reactions to these agents included severe renal insufficiency, hyponatremia, and neutropenia. The doses of ACV administered via continuous infusion ranged from 30 to 50 mg/kg per day, and time to resolution of lesions ranged from 1 week to 2 months. No patients experienced toxicity associated with continuous infusion of high dose acyclovir and therapy could be continued as an outpatient when appropriate.Age, sexDiseaseHCT TypeConcomitant risk factorsHSV Serotype, locationMIC50(mcg/mL)Failed HSV Treatment Regimens***High-dose continuous ACV (mg/kg/d)Outcome, Time to responseGVHDPREDOtherACVGCVFOS37, FAMLTCD URD MASkin, Grade 2 GI<0.5 mg/k/dCSAHSV-2; genitalia102222ACV IV FOS IV CID IV30Resolved; 3 weeks47, FMDS, AMLURD MASkin, Grade 4<0.5-2 mg/k/dFK506 MMFHSV-2; genitalia, buttocks, upper thighs>48>48>200ACV IV FOS IV45Resolved; 2 months55, FMDSURD MASkin, Grade 250 mg qdTAC MMFHSV-1; Oro-pharynx>48>48>48ACV IV FOS IV30Resolved; 1 week44, FET, AMLURD MASkin, Grade 460 mg qdPrografHSV-1; Tongue>48>48>48ACV IV FAM PO30 - 50Partially resolved***24, MATMRD MANoneNoneCSA ATGHSV-1 Nare>48>48>100ACV PO FAM PO30Resolved; 1 week*All pts received allogeneic transplants and HSV prophylactic regimen of acyclovir**Dose ranges: Acyclovir 400 mg po 5x qd to 10 mg/kg IV q 8 hrs; cidofovir 5 mg/kg q week; foscarnet 40 mg/kg qd to 25 mg/kg q 12 hrs; famciclovir 500 mg po bid to 1000 mg po tid***Patient expired prior to resolution of lesions MDS = myelodysplastic syndrome; AML = acute myelogenous leukemia; URD = unrelated donor; MRD = matched-related donor; TCD = T-cell depleted; MA = myeloablative; ET = Essential thrombocytosis, AT = Amegakarocytic thrombocytopenia PRED = prednisone PO ; ACV = acyclovir, GCV = ganciclovir; FOS = foscarnet; CID = cidofovir; FAM = famciclovir; TAC = tacrolimus Conclusions:Continuous infusion of high dose acyclovir for acyclovir resistant HSV is an attractive and more tolerable alternative for patients failing standard dose intermittent infusion acyclovir or intolerant of alternative standard therapies such as foscarnet, cidofovir, or ganciclovir. At this time it is not clear how the altered pharmacokinetics that results from continuous infusion of high doses of acyclovir overcomes viral resistance. We are currently investigating the mechanisms by which the sustained levels of ACV provide this therapeutic advantage. Disclosures:No relevant conflicts of interest to declare.

Antiviral activity of Cleome rosea extracts from field-grown plants and tissue culture-derived materials against acyclovir-resistant Herpes simplex viruses type 1 (ACVr-HSV-1) and type 2 (ACVr-HSV-2)

Antiviral activity of Cleome rosea extracts from field-grown plants and tissue culture-derived materials against acyclovir-resistant Herpes simplex viruses type 1 (ACVr-HSV-1) and type 2 (ACVr-HSV-2)

Antiviral activity of Cleome rosea extracts from field-grown plants and tissue culture-derived materials against acyclovir-resistant Herpes simplex viruses type 1 (ACVr-HSV-1) and type 2 (ACVr-HSV-2)

Extracts from Cleome rosea were investigated for their activity against acyclovir-resistant strains of Herpes simplex type 1 (ACVr-HSV-1) and type 2 (ACVr-HSV-2). Methanolic and acidified (1% (v/v) HCl) methanolic extracts were prepared from field-grown plants and in vitro propagated plants, as well as from calli and cell suspension cultures. The extracts presented low cytotoxicity and caused virus titer reduction above 70%, with different mechanisms of action. Extracts from leaves of field-grown plants inhibited viral infection mainly by affecting the virus particle itself (virucidal effect), while extracts from calli acted mainly on cell receptors. On the other hand, all extracts evaluated affected the virus entry across the cell membrane and the intracellular viral replication at similar percentages, causing reduction on titers in the range of 68–90%. This study validated the potential use of in vitro materials as sources of antiherpetic agents from C. rosea.

Impact of Ethanolic Lamiaceae Extracts on Herpesvirus Infectivity in Cell Culture

Background: Extracts of medicinal plants are increasingly of interest as novel drugs for antimicrobial and antiviral agents, since microorganisms might develop resistance to commonly used antimicrobial or antiviral agents. Materials and Methods: Ethanolic extracts from Lamiaceae plants prunella, peppermint, rosemary and thyme were phytochemically characterised. The inhibitory activity of four 20% ethanolic plant extracts and four 80% ethanolic extracts against herpes simplex virus (HSV) strains was tested in cell culture. Results: Rosmarinic acid, a typical compound in Lamiaceae species, was identified in the extracts except for thyme 20% ethanolic extract. In addition, some other phenolic compounds such as apigenin- and luteolin-derivatives were identified in different amounts. All extracts exhibited high and concentration-dependent levels of antiviral activity against free acyclovir-sensitive and acyclovir-resistant HSV-1 strains with 50% inhibitory concentrations of 0.05–0.82 μg/ml. Mechanistically, exposure of free virions as well as host cells to prunella and peppermint 80% ethanolic extracts at maximum non-cytotoxic concentrations prior to infection reduced plaque formation drastically. Thus, both extracts revealed a dual mode of action similar to aqueous lemon balm extracts. Conclusions: Since infectivity of acyclovir-susceptible and acyclovir-resistant HSV strains was significantly reduced with Lamiaceae extracts, the results obtained indicate that ethanolic plant extracts affected herpesvirus prior to and during adsorption and in a different way than acyclovir. Based on its dual mode of action, e.g. antiviral effect against free virions and blocking virus attachment to host cells, prunella and peppermint 80% ethanolic extracts are promising antiviral agents in recurrent herpes labialis for topical therapeutic applications.

Chronic Hand Ulcer in a Woman With HIV Infection

CASE PRESENTATION A 23-year-old female resident of an AIDS hospice in Ho Chi Minh City had a chronic ulcer on her left hand (Fig. 1). The lesion started on the tip of the third finger and over the course of 1 year slowly progressed to include the ventral palm. On examination, the ulcer was well circumscribed with mild surrounding edema and tenderness. There was no bleeding or purulence. Sensation in the unaffected areas was normal. Motor function of the hand was normal. She had similar lesions on her buttocks, which had also appeared around the same time as the finger ulcer. She had been diagnosed as HIV positive 3 years earlier. CD4 testing was not available, and she had never taken antiretroviral medications. The patient had no history of fever, weight loss, cough, or other systemic symptoms except for intermittent diarrhea. She was single and had previously sold seafood in a local market, which involved regularly handling live fish.FIGURE 1: Chronic left-hand ulcer.What is your diagnosis? Differential diagnosis: Cutaneous tuberculosis or atypical mycobacterium, cutaneous fungal infection, herpes simplex virus (HSV), varicella-zoster virus, malignancy, bacterial infection. Diagnosis: Herpes simplex virus infection DISCUSSION Complete blood count and liver function tests were within normal limits. VDRL was negative. The pathology reading on a biopsy performed at local hospital was reported as "nonspecific inflammation" with negative acid-fast bacillus and fungal stains. The patient had taken multiple courses of antibiotic and antifungal medications without improvement. The patient was treated empirically with acyclovir 200 mg 5 times a day, and within 2 weeks, the ulcers had almost completely healed (Fig. 2). After 4 weeks of treatment, the dose of acyclovir was reduced to 200 mg 3 times a day. Three months later, the patient left the hospice and was lost to follow-up.FIGURE 2: Left hand after 2 weeks' treatment with acyclovir.The diagnosis of HSV infection can usually be made by viral culture or direct fluorescent antibody testing. A Tzanck preparation from direct smear of the lesion can help to narrow the differential to herpesviruses. Polymerase chain reaction for HSV DNA is sensitive, but the test has not been FDA cleared for use on genital lesions.1 Serological testing for HSV antibodies is available, but only the newer glycoprotein G-based type-specific assays can differentiate between HSV-1 and HSV-2. Testing for HSV was not available in Vietnam at the time this patient was treated. A presumptive diagnosis of a herpesvirus infection was made based on the rapid response of the ulcer to treatment with acyclovir. More than 80% of adults in Asia have evidence of previous HSV-1 infection by antibody testing.2 The prevalence of HSV-2 infection is lower in Asian countries than in African and western nations.3 There are little data on HSV infection rates in Vietnam, but 1 study of female sex workers in 3 Mekong Delta provinces showed HSV-2 antibody prevalence rates of 30% to 33%.4 Atypical presentations of herpes are not uncommon in immunocompromised patients.4 Ulcers can appear in single or multiple locations and are more likely to become chronic and extensive. Herpes simplex virus lesions in the HIV-infected patient can usually be treated with acyclovir, although acyclovir-resistant HSV has been reported.5,6 Severe or disseminated HSV infections may require treatment with high-dose intravenous acyclovir. In the immunocompromised patient, chronic ulcers may be due to infectious causes (bacterial, mycobacterial, fungal, or viral) or noninfectious causes such as malignancy. Opportunistic infections of the hand due to Cryptococcus and Candida have been reported.6 Ulcers can also become secondarily infected with bacteria, making the underlying primary etiology more difficult to diagnose. Varicella-zoster virus is another herpesvirus that can cause skin disease in immunocompromised hosts. The lesions usually appear as clusters of vesicles localized to one or more dermatomes, but can coalesce into large ulcers or become disseminated. Varicella-zoster virus may also respond to treatment with acyclovir, but generally requires higher doses than are used for treatment of HSV disease. This patient had a history of regular live fish exposure, occasionally associated with bacterial infections such as Erysipelothrix rhusiopathiae or Mycobacterium marinum. In this case, the acid-fast bacillus stain on the biopsy was negative, and the diagnosis of HSV infection was made by the rapid and complete response to an empiric trial of acyclovir. Initial management of ulcerative diseases in HIV patients should include microbiological evaluation of the lesion. Biopsy may be indicated when microbiological evaluation is nondiagnostic. In resource-limited settings where laboratory capabilities are limited, however, empiric treatment of chronic ulcers with antiviral medication active against HSV may be both diagnostic and curative.

Genotypic detection of acyclovir-resistant HSV-1: Characterization of 67 ACV-sensitive and 14 ACV-resistant viruses

Infections due to herpes simplex virus (HSV) resistant to acyclovir (ACV) represent an important clinical concern in immunocompromised patients. In order to switch promptly to an appropriate treatment, rapid viral susceptibility assays are required. We developed herein a genotyping analysis focusing on thymidine kinase gene (TK) mutations in order to detect acyclovir-resistant HSV in clinical specimens. A total of 85 HSV-1 positive specimens collected from 69 patients were analyzed. TK gene could be sequenced directly for 81 clinical specimens (95%) and 68 HSV-1 specimens could be characterized as sensitive or resistant by genotyping (84%). Genetic characterization of 67 susceptible HSV-1 specimens revealed 10 polymorphisms never previously described. Genetic characterization of 14 resistant HSV-1 revealed 12 HSV-1 with either TK gene additions/deletions (8 strains) or substitutions (4 strains) and 2 HSV-1 with no mutation in the TK gene. DNA polymerase gene was afterwards explored. With this rapid PCR-based assay, ACV-resistant HSV could be detected directly in clinical specimens within 24 h.

Frequency of acyclovir‐resistant herpes simplex viruses isolated from the general immunocompetent population and patients with acquired immunodeficiency syndrome

Herpes simplex virus (HSV) infections are usually chronically recurrent in the normal population and represent a significant cause of morbidity in immunocompromised patients. Acyclovir (ACV) is widely used for the treatment and prophylaxis of HSV infections. The emergence of ACV-resistant strains has been frequently reported as a result of long-term ACV therapy. Despite the widespread use of ACV, there are no data available in our area on the frequency of ACV-resistant HSVs. The purpose of this study was to evaluate the susceptibility of HSV isolated from normal subjects and patients with acquired immunodeficiency syndrome (AIDS) to ACV. HSVs were isolated from the orofacial region of normal individuals and patients with AIDS. The susceptibility of isolated HSV strains to various concentrations of ACV was determined by plaque reduction assay. The sensitivity of the viral strains was expressed as IC(50) (the concentration of drug reducing the viral plaque by 50%). One hundred and thirty-three isolates from 102 normal subjects and 31 patients with AIDS were tested. One HSV-1 isolate from normal individuals had intermediate susceptibility. Two ACV-resistant isolates (one HSV-1 and one HSV-2), with IC(50) > or = 2 to < or = 3 microg/mL, and one highly resistant HSV-2 isolate, with IC(50) > or = 5 microg/mL, were detected in patients with AIDS. Our data show that the prevalence of ACV-resistant strains is very low in the general immunocompetent population; however, in patients with AIDS, the prevalence of ACV-resistant strains is remarkable (P = 0.001). Alternative antiherpetic agents should be employed to control and reduce the emergence of ACV-resistant strains in patients with AIDS.

Use of Long‐Term Suppressive Acyclovir after Hematopoietic Stem‐Cell Transplantation: Impact on Herpes Simplex Virus (HSV) Disease and Drug‐Resistant HSV Disease

The effect that long-term use of suppressive acyclovir (ACV) has on both overall herpes simplex virus (HSV) disease and ACV-resistant HSV disease was examined in 3 consecutive cohorts of hematopoietic stem-cell transplant (HCT) recipients (n=2049); cohort 1 received ACV for 30 days after HCT, cohort 2 received it for 1 year after HCT, and cohort 3 received it for an extended period (i.e., >1 year) if the patient's immunosuppression continued after 1 year. The 2-year probability of HSV disease was 31.6% (95% confidence interval [CI], 28.0%-35%) in cohort 1, 3.9% (95% CI, 2.7%-5.2%) in cohort 2, and 0% in cohort 3 (P<.001). ACV-resistant HSV disease developed in 10 patients in cohort 1 (2-year probability, 1.3% [95% CI, 0.8%-2.7%]), in 2 patients in cohort 2 (2-year probability, 0.2% [95% CI, 0%-0.8%]; P=.006), and in 0 patients in cohort 3 (cohort 2 vs. cohort 3, P=.3). Long-term use of suppressive prophylactic ACV appears to prevent the emergence of drug-resistant HSV disease in HCT.

Expression of Extremely Low Levels of Thymidine Kinase from an Acyclovir-Resistant Herpes Simplex Virus Mutant Supports Reactivation from Latently Infected Mouse Trigeminal Ganglia

A single-cytosine-deletion in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. A laboratory strain engineered to carry this mutation did not generate sufficient TK activity for detection by plaque autoradiography, which detected 0.25% wild-type activity. However, a drug sensitivity assay suggested that extremely low levels of TK are generated by this virus. The virus was estimated to express 0.09% of wild-type TK activity via a ribosomal frameshift 24 nucleotides upstream of the mutation. Remarkably, this appeared to be sufficient active TK to support a low level of reactivation from latently infected mouse trigeminal ganglia.

Dendritic Keratitis Caused by an Acyclovir-Resistant Herpes Simplex Virus With Frameshift Mutation

To report a case of acyclovir-resistant herpes simplex virus (HSV) keratitis after long-term, inconsistent use of topical acyclovir and fluorometholone. A 70-year-old man with dendritic keratitis caused by an acyclovir-resistant HSV strain was examined. The 50% inhibitory concentration of different antiviral agents against the isolated virus and the DNA sequence of viral thymidine kinase were determined. The 50% inhibitory concentration of acyclovir and trifluorothymidine for the isolated HSV strain was 13.75 and 0.28 microg/mL, respectively, indicating that the virus was resistant to acyclovir. DNA sequencing of the viral thymidine kinase revealed that this virus had a frameshift mutant with a G insertion in the 7Gs homopolymer. Topical trifluorothymidine was effective, and the epithelial lesion was completely resolved within 2 weeks. A typical form of dendritic keratitis was caused by an acyclovir-resistant HSV with frameshift mutation in a 7Gs homopolymer region.

Selective Excision of Chain-terminating Nucleotides by HIV-1 Reverse Transcriptase with Phosphonoformate as Substrate

A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3'-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP(i)) analogs on PP(i)- and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP(i)- and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT.template/primer complex was hindered by the presence of a chain terminator at the 3'-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZT-triphosphate in the presence of PP(i) or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.

Pathogenicity and response to topical antiviral therapy in a murine model of acyclovir-sensitive and acyclovir-resistant herpes simplex viruses isolated from the same patient

Background Acyclovir-resistant herpes simplex viruses (HSV) are commonly recovered from immunocompromised individuals who exhibit chronic and/or disseminated herpetic lesions. Objectives and study design The virulence and response to topical acyclovir therapy were evaluated in a mouse model of zosteriform cutaneous HSV infection using two HSV-1 isolates from the same immunocompromised patient (one susceptible and one resistant to acyclovir). Results The acyclovir-resistant virus, with a Thr63Ile change in the ATP-binding site of the thymidine kinase gene, produced almost as many skin lesions as the wild-type susceptible virus. As expected from in vitro susceptibility data, the herpetic lesions of the mice infected with the drug-resistant virus did not respond to topical acyclovir therapy. Conclusions Some thymidine kinase HSV mutants associated with drug resistance may retain their pathogenicity at least in the mouse model described in this study.