Jeffry J. Leary

Resistance of Herpes Simplex Virus Infections to Nucleoside Analogues in HIV-Infected Patients

Antiviral treatment of herpes simplex virus (HSV) infections with nucleoside analogues has been well established for >2 decades, but isolation of drug-resistant HSV from immunocompetent patients has remained infrequent (0.1%-0.7% of isolates) during this period. Even when drug-resistant HSV is isolated from an immunocompetent patient, this virus, with rare exceptions, is cleared normally without adverse clinical outcome. Although drug-resistant HSV is more commonly isolated from immunocompromised patients (4%-7% of isolates) and is more likely to be clinically significant, the prevalence of drug-resistant HSV even among these patients, has been stable over the past 2 decades. Despite this stable prevalence, disease due to drug-resistant HSV remains an important problem for many immunocompromised patients, including those with HIV infection. This article reviews the prevalence, pathogenesis, and implications of drug-resistant HSV infections in HIV-infected patients.

Characterization of Herpes Simplex Viruses Selected in Culture for Resistance to Penciclovir or Acyclovir

ABSTRACT Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acvr clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcvr mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.

Difference in Incidence of Spontaneous Mutations between Herpes Simplex Virus Types 1 and 2

ABSTRACT Spontaneous mutations within the herpes simplex virus (HSV) genome are introduced by errors during DNA replication. Indicative of the inherent mutation rate of HSV DNA replication, heterogeneous HSV populations containing both acyclovir (ACV)-resistant and ACV-sensitive viruses occur naturally in both clinical isolates and laboratory stocks. Wild-type, laboratory-adapted HSV type 1 (HSV-1) strains KOS and Cl101 reportedly accumulate spontaneous ACV-resistant mutations at a frequency of approximately six to eight mutants per 104plaque-forming viruses (U. B. Dasgupta and W. C. Summers, Proc. Natl. Acad. Sci. USA 75:2378–2381, 1978; J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. T. Gelep, and P. A. Schaffer, Virology 132:26–37, 1984). Typically, these resistance mutations map to the thymidine kinase (TK) gene and render the virus TK deficient. To examine this process more closely, a plating efficiency assay was used to determine whether the frequencies of naturally occurring mutations in populations of the laboratory strains HSV-1 SC16, HSV-2 SB5, and HSV-2 333 grown in MRC-5 cells were similar when scored for resistance to penciclovir (PCV) and ACV. Our results indicate that (i) HSV mutants resistant to PCV and those resistant to ACV accumulate at approximately equal frequencies during replication in cell culture, (ii) the spontaneous mutation frequency for the HSV-1 strain SC16 is similar to that previously reported for HSV-1 laboratory strains KOS and Cl101, and (iii) spontaneous mutations in the laboratory HSV-2 strains examined were 9- to 16-fold more frequent than those in the HSV-1 strain SC16. These observations were confirmed and extended for a group of eight clinical isolates in which the HSV-2 mutation frequency was approximately 30 times higher than that for HSV-1 isolates. In conclusion, our results indicate that the frequencies of naturally occurring, or spontaneous, HSV mutants resistant to PCV and those resistant to ACV are similar. However, HSV-2 strains may have a greater propensity to generate drug-resistant mutants than do HSV-1 strains.

Frequency of acyclovir-resistant herpes simplex virus in clinical specimens and laboratory isolates

ABSTRACT The proportion of acyclovir (ACV)-resistant herpes simplex virus (HSV) isolates in clinical specimens and laboratory isolates was determined. HSV isolates in clinical specimens and laboratory isolates were cultured in the absence or presence of 5 μg of ACV per ml. The frequency of ACV-resistant HSV was calculated as (average virus titer in the wells with ACV)/(average virus titer in the wells without ACV). The mutation frequency of HSV type 1 isolates in clinical samples (directly from patient lesions) was 7.5 × 10−4± 2.5 × 10−4 (mean ± standard error), and that of HSV type 2 isolates was 15.0 × 10−4 ± 4.6 × 10−4. The mutation frequencies of isolates derived in the laboratory from these clinical samples were very similar. Similarly, the 50% inhibitory concentrations for isolates in clinical samples and laboratory isolates were identical. The frequencies of ACV-resistant HSV types 1 and 2 were in a narrow range of 7.5 × 10−4 to 15.0 × 10−4 among isolates in clinical specimens and did not change for laboratory-derived pools of viral isolates.

Susceptibilities of Herpes Simplex Viruses to Penciclovir and Acyclovir in Eight Cell Lines

ABSTRACT The commonly used antiviral drugs acyclovir (ACV) and penciclovir (PCV) possess similarly potent antiviral activities in vivo against herpes simplex virus (HSV). Assay methods for sensitivity to ACV are not necessarily transferable to PCV, even though the two drugs have similar in vivo potencies and mechanisms of action. We determined by plaque reduction assay the relative activities of ACV and PCV against five laboratory-adapted strains of HSV types 1 and 2 (including sensitive and resistant strains) in seven human cell lines and one nonhuman primate cell line. Seven characteristics were used to evaluate the cell lines. All cell lines were similar in their plating efficiencies and abilities to discriminate between sensitive and resistant HSV isolates. Vero and MRC-5 cells yielded the most discordant 50% inhibitory concentrations (IC50s) for the two HSV types, while Vero and WI-38 VA-13 cells yielded large differences in the IC50s of ACV and PCV. The limited life spans and poor plaque morphologies of the fibroblast lines were undesirable characteristics. Among the transformed cell lines producing well-defined plaques, A549 cells provided the best concordance between IC50s for the two HSV types and two antiherpes drugs. Comparison experiments with a yield reduction format indicated that the use of assays of this type might allow some of the cell-specific properties observed in plaque reduction assays to be avoided.

Development of Acyclovir-resistant herpes simplex virus early during the treatment of herpes neonatorum

Genotypic analysis of herpes simplex virus (HSV) DNA extracted from clinical specimens from a case of fatal disseminated neonatal HSV demonstrated that an infant developed an acyclovir-resistant HSV containing a mutation in the HSV thymidine kinase gene during the first seven days of acyclovir therapy.

GSK983: a novel compound with broad-spectrum antiviral activity.

Abstract GSK983, a novel tetrahydrocarbazole, inhibits the replication of a variety of unrelated viruses in vitro with EC50 values of 5–20nM. Both replication of the adenovirus Ad-5 and the polyoma virus SV-40, and episomal maintenance of human papillomaviruses (HPV) and Epstein-Barr virus (EBV) are susceptible to GSK983. The compound does not inhibit all viruses; herpes simplex virus (HSV-1), human immunodeficiency virus (HIV), and lytic replication of EBV were not susceptible at concentrations below 1μM. GSK983 does inhibit the growth of cell lines immortalized by HTLV-1, EBV, HPV, SV40 and Ad-5, with EC50 values in the range of 10–40nM. Depending on the cell line, the compound induces either apoptosis or cytostasis at concentrations over 20nM. GSK983 also inhibits cell lines immortalized by non-viral mechanisms, but has little effect on primary cells. The CC50 values for keratinocytes, fibroblasts, lymphocytes, endothelial, and bone marrow progenitor cells are all above 10μM. The pattern of inhibition, which includes diverse viruses as well as growth of immortalized cells of varied origins, suggests the target is a host cell protein, rather than a viral protein. Preliminary mechanism studies indicate that GSK983 acts by inducing a subset of interferon-stimulated genes.

Effects of Antiviral Usage on Transmission Dynamics of Herpes Simplex Virus Type 1 and on Antiviral Resistance: Predictions of Mathematical Models

ABSTRACT Herpes simplex virus type 1 (HSV-1) causes recurrent herpes labialis (RHL), a common disease afflicting up to 40% of adults worldwide. Mathematical models are used to analyze the effect of antiviral treatment on the transmission of, and the prevalence of drug resistance in, HSV-1 in the United States. Three scenarios are analyzed: no antiviral use, the current level of use, and a substantial increase in nucleoside analogue use, such as might occur if topical penciclovir were available over-the-counter for the treatment of RHL. A basic model predicts that present level of nucleoside analogue use has a negligible effect on HSV-1 transmission and that even if use of topical penciclovir for (RHL) increased substantially, the overall prevalence of infectious HSV-1 is unlikely to be reduced by more than 5%. An expanded model, which allows for acquired resistance and includes immunocompromised hosts and other more realistic features, predicts that current antiviral use is unlikely to lead to any noticeable increase in resistance. If antiviral use increases, the resulting rise in resistance in the population will depend primarily on the probability that immunocompetent hosts will acquire permanent resistance upon treatment. This probability is known to be small, but its exact value remains uncertain. If acquired resistance occurs less than once per 2,500 treated episodes, then in the community at large, the frequency of HSV-1 resistance is predicted to increase slowly, if at all (remaining below 0.5% for >50 years), even with extensive nucleoside analogue use. If acquired resistance emerges in 1 of 625 treated episodes (the maximum of an approximate 95% confidence interval derived from the results of several studies of resistance in treated hosts), then the prevalence of infection with resistant HSV-1 could rise from about 0.2% to 1.5 to 3% within 50 years. The limitations of existing data on acquired resistance and the potential impact of acquired resistance if it occurs are discussed, and strategies are suggested for enhancing information on acquired resistance. The predictions of this model contrast with the more rapid increases in antimicrobial resistance anticipated by models and observed for other pathogenic bacteria and viruses. The reasons for these contrasting predictions are discussed.

Penciclovir Susceptibilities of Herpes Simplex Virus Isolates from Patients Using Penciclovir Cream for Treatment of Recurrent Herpes Labialis

ABSTRACT The antiherpesvirus agent penciclovir (PCV) shares an identical activation pathway and a similar mode of action with acyclovir (ACV). However, since PCV represents a relatively recent treatment option, the clinical resistance profile to PCV is less well known. A susceptibility program was established to assess the resistance profile for serial herpes simplex virus isolates from immunocompetent patients with recurrent herpes labialis obtained throughout a 4-day period of treatment with topical PCV (1% cream) or a placebo. Two isolates (2 of 1,035 [0.19%]), representing 0.34% of the patients (2 of 585), were confirmed to be PCV-resistant (Pcvr) herpes simplex virus type 1 by a plaque reduction assay in MRC-5 cells. These two viruses were highly resistant to PCV (50% inhibitory concentrations [IC50s], >55 μg/ml) and were isolated less than 17 h after the start of patient-initiated treatment. However, subsequent isolates on days 2 and 3 from these patients were completely susceptible to PCV (IC50s, <2.0 μg/ml). Thus, it is not clear whether the resistance to PCV for these two early-treatment isolates was directly associated with the 17 h of PCV treatment; several possible explanations are discussed. In an analysis of the distribution of IC50 differences between the first and last isolates, there were three patients with minor IC50 increases in the PCV-treated population and one in the placebo-treated group, although statistically, only the latter was an outlier. No patients were found to have Pcvr virus at the end of acute treatment, regardless of treatment group. Overall, the prevalence of Pcvr was found to be similar to the 0.3% Acvr reported for immunocompetent, untreated populations.

Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations

BackgroundThe thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1.MethodsA panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases.ResultsAlthough HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture.ConclusionsThis study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.