Julie Bestman-Smith

Resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms

Nucleoside analogues such as acyclovir and ganciclovir have been the mainstay of therapy for alphaherpesviruses (herpes simplex virus (HSV) and varicella-zoster virus (VZV)) and cytomegalovirus (CMV) infections, respectively. Drug-resistant herpesviruses are found relatively frequently in the clinic, almost exclusively among severely immunocompromised patients receiving prolonged antiviral therapy. For instance, close to 10% of patients with AIDS receiving intravenous ganciclovir for 3 months excrete a drug-resistant CMV isolate in their blood or urine and this percentage increases with cumulative drug exposure. Many studies have reported that at least some of the drug-resistant herpesviruses retain their pathogenicity and can be associated with progressive or relapsing disease. Viral mutations conferring resistance to nucleoside analogues have been found in either the drug activating/phosphorylating genes (HSV or VZV thymidine kinase, CMV UL97 kinase) and/or in conserved regions of the viral DNA polymerase. Currently available second line agents for the treatment of herpesvirus infections--the pyrophosphate analogue foscarnet and the acyclic nucleoside phosphonate derivative cidofovir--also inhibit the viral DNA polymerase but are not dependent on prior viral-specific activation. Hence, viral DNA polymerase mutations may lead to a variety of drug resistance patterns which are not totally predictable at the moment due to insufficient information on specific drug binding sites on the polymerase. Although some CMV and HSV DNA polymerase mutants have been found to replicate less efficiently in cell cultures, further research is needed to correlate viral fitness and clinical outcome.

Drug Resistance Patterns of Recombinant Herpes Simplex Virus DNA Polymerase Mutants Generated with a Set of Overlapping Cosmids and Plasmids

ABSTRACT Herpes simplex virus (HSV) DNA polymerase (Pol) mutations can confer resistance to all currently available antiherpetic drugs. However, discrimination between mutations responsible for drug resistance and those that are part of viral polymorphism can be difficult with current methodologies. A new system is reported for rapid generation of recombinant HSV type 1 (HSV-1) DNA Pol mutants based on transfection of a set of overlapping viral cosmids and plasmids. With this approach, twenty HSV-1 recombinants with single or dual mutations within the DNA pol gene were successfully generated and subsequently evaluated for their susceptibilities to acyclovir (ACV), foscarnet (FOS), cidofovir (CDV), and adefovir (ADV). Mutations within DNA Pol conserved regions II (A719T and S724N), VI (L778M, D780N, and L782I), and I (F891C) were shown to induce cross-resistance to ACV, FOS, and ADV, with two of these mutations (S724N and L778M) also conferring significant reduction in CDV susceptibility. Mutant F891C was associated with the highest levels of resistance towards ACV and FOS and was strongly impaired in its replication capacity. One mutation (D907V) lying outside of the conserved regions was also associated with this ACV-, FOS-, and ADV-resistant phenotype. Some mutations (K522E and Y577H) within the δ-region C were lethal, whereas others (P561S and V573M) induced no resistance to any of the drugs tested. Recombinants harboring mutations within conserved regions V (N961K) and VII (Y941H) were resistant to ACV but susceptible to FOS. Finally, mutations within conserved region III were associated with various susceptibility profiles. This new system allows a rapid and accurate evaluation of the functional role of various DNA Pol mutations, which should translate into improved management of drug-resistant HSV infections.

Highly Reliable Heterologous System for Evaluating Resistance of Clinical Herpes Simplex Virus Isolates to Nucleoside Analogues

ABSTRACT Clinical resistance of herpes simplex virus (HSV) types 1 and 2 to acyclovir (ACV) is usually caused by the presence of point mutations within the coding region of the viral thymidine kinase (TK) gene. The distinction between viral TK mutations involved in ACV resistance or part of viral polymorphism can be difficult to evaluate with current methodologies based on transfection and homologous recombination. We have developed and validated a new heterologous system based on the expression of the viral TK gene by the protozoan parasiteLeishmania, normally devoid of TK activity. The viral TK genes from 5 ACV-susceptible and 13 ACV-resistant clinical HSV isolates and from the reference strains MS2 (type 2) and KOS (type 1) were transfected as part of an episomal expression vector inLeishmania. The susceptibility of TK-recombinant parasites to ganciclovir (GCV), a closely related nucleoside analogue, was evaluated by a simple measurement of the absorbance ofLeishmania cultures grown in the presence of the drug. Expression of the TK gene from ACV-susceptible clinical isolates resulted in Leishmania susceptibility to GCV, whereas expression of a TK gene with frameshift mutations or nucleotide substitutions from ACV-resistant isolates gave rise to parasites with high levels of GCV resistance. The expression of the HSV TK gene inLeishmania provides an easy, reliable, and sensitive assay for evaluating HSV susceptibility to nucleoside analogues and for assessing the role of specific viral TK mutations.

Sodium Lauryl Sulfate Abrogates Human Immunodeficiency Virus Infectivity by Affecting Viral Attachment

ABSTRACT The microbicidal activity of sodium lauryl sulfate (SLS) against human immunodeficiency virus type 1 (HIV-1) was studied in cultured cells. Pretreatment of HIV-1NL4-3 with SLS decreased, in a concentration-dependent manner, its infectivity when using 1G5 as target cells. In the absence of a viral pretreatment period or when 1G5 cells were pretreated with SLS, the surfactant-induced inactivation of viral infectivity was less pronounced, especially at concentrations between 375 and 550 μM. SLS had no effect on HIV-1 when the virus was adsorbed to 1G5 cells by a 2-h incubation period. SLS almost completely inhibited the fusion process by decreasing the attachment of HIV-1 to target cells. SLS also inhibited the infectivity of HIV-1-based luciferase reporter viruses pseudotyped with the amphotropic murine leukemia virus envelope (which enters cells in a CD4-, CCR5-, and CXCR4-independent manner), indicating that SLS may inactivate other envelope viruses. In contrast, no effect was seen with vesicular stomatitis virus envelope glycoprotein G (which enters cells through receptor-mediated endocytosis) pretreated with up to 700 μM SLS. SLS also decreased, in a dose-dependent manner, the HIV-1-dependent syncytium formation between 1G5 and J1.1 cells after a 24-h incubation. The reduction of luciferase activity was more pronounced when J1.1 cells (which express HIV-1 proteins on their surface) were pretreated with SLS rather than 1G5 cells. Taken together, our results suggest that SLS could represent a candidate of choice for use in vaginal microbicides to prevent the sexual transmission of HIV and possibly other pathogens causing sexually transmitted diseases.

Targeting cell-free HIV and virally-infected cells with anti-HLA-DR immunoliposomes containing amphotericin B

ObjectiveTo evaluate the ability of liposomes bearing anti-HLA-DR Fab′ fragments (immunoliposomes) and containing amphotericin B (AmB) to target and neutralize cell-free HIV-1 particles and virally-infected cells. MethodsThe effect of AmB on the attachment and fusion of HIV-1NL4−3to Jurkat E6.1 cells has been evaluated using a p24 enzymatic assay. The ability of AmB to inhibit HIV-1-based luciferase reporter viruses pseudotyped with HXB2, AML-V and VSV-G envelopes has been evaluated in Jurkat E6.1 cells. The efficacy of free and immunoliposomal AmB to inhibit cell-free HIV, that have incorporated or not HLA-DR molecules, has been evaluated in HLA-DR/negative (NEG) 1G5 T cells and HLA-DR/positive (POS) Mono Mac 1 cells. ResultsAmB inhibited HIV infectivity independently of the nature of viral envelope proteins. Pretreatment of HIV with AmB had no major effect on viral attachment and fusion process to Jurkat E6.1 cells. Immunoliposomal AmB (0.5 μg/ml) led to a 77% inhibition of replication of HLA-DR/POS HIV-1 with no cell toxicity, whereas free AmB had no significant antiviral activity at this concentration. A complete inhibition of viral replication was observed following incubation of viruses with immunoliposomal AmB (2.5 μg/ml). Anti-HLA-DR immunoliposomes containing AmB had no effect on the infectivity of HLA-DR/NEG HIV-1 particles in HLA-DR/NEG T lymphoid cells but completely inhibited replication of viruses in an HLA-DR/POS monocytic cell line. ConclusionThe incorporation of neutralizing agents in anti-HLA-DR immunoliposomes could represent a novel therapeutic strategy to specifically target cell-free HIV particles and virally-infected cells to treat HIV infection more efficiently.

A Cluster of Three Cases of Hantavirus Pulmonary Syndrome among Canadian Military Personnel

Hantavirus pulmonary syndrome (HPS) is a rare illness in eastern Canada. We present three cases of HPS among military personnel in Quebec. The three cases shared a common exposure to mouse excreta while engaged in military training in Alberta, a western province of Canada.