Absence of rapid selection for acyclovir or penciclovir resistance following suboptimal oral prodrug therapy of HSV-infected mice.

BACKGROUND: Acyclovir (ACV) resistant herpes simplex virus (HSV) isolates can be readily selected in animal infection models receiving suboptimal ACV treatment, however no comparative studies of the emergence of resistance following suboptimal treatment with valacyclovir (VCV) or famciclovir (FCV), the prodrugs of acyclovir and penciclovir, respectively, have been reported. METHODS: Mice (n = 30) were infected with HSV type 1 or 2 in the ear pinnae and administered oral prodrugs at one fifth a dose previously shown to be effective. To select and amplify drug-resistant HSV, a total of seven consecutive in vivo passages with suboptimal treatment were performed for each virus sample and progeny virus from each passage was characterized by the plaque reduction (PRA) and plating efficiency assays (PEA). RESULTS: No drug-resistant HSV-2 and only a single drug-resistant HSV-1 variant were identified. Virus recovered from the first three sequential passages of this HSV-1 sample was susceptible by PRA, although the proportion of resistant virus recovered gradually increased upon passage. The resistant HSV-1 phenotype was confirmed by PRA after four sequential passages in mice. Unexpectedly, this in vivo-selected drug-resistant HSV-1 failed to yield an infection completely refractory to treatment in subsequent passages. CONCLUSIONS: Sub-optimal therapy of immunocompetent mice with either VCV or FCV did not readily select for HSV-mutants resistant to either ACV or PCV, suggesting that selection of resistance with either prodrug remains difficult using this system. Futhermore, this study suggests that the PEA may represent a useful adjunct to the PRA for monitoring alterations in the proportion of drug-resistant virus even when no change in IC50 is apparent.

Biochemical characterization of a virus isolate, recovered from a patient with herpes keratitis, that was clinically resistant to acyclovir.

In vitro susceptibility assays of herpes simplex virus (HSV) do not necessarily correlate with treatment outcome. An HSV type 1 (HSV-1) isolate, N4, recovered from a patient who presented with herpes keratitis with localized immunosuppression, was characterized for susceptibility. Although the 50% inhibitory concentration (IC(50)) for this isolate was less than the accepted breakpoint for defining resistance to acyclovir (>2.0 microg/mL), the following lines of evidence suggest that the isolate was acyclovir resistant: (1) the clinical history confirmed that the infection was nonresponsive to acyclovir; (2) the in vitro susceptibility was similar to that of a thymidine kinase (TK)-negative, acyclovir-resistant virus SLU360; (3) the IC(50) of acyclovir was more than 10 times the IC(50) for an acyclovir-susceptible control strain; (4) plaque-purified clonal isolates were resistant to acyclovir (IC(50)s, >2.0 microg/mL); and (5) biochemical studies indicated that the HSV-1 N4 TK was partially impaired for acyclovir phosphorylation. Although residue changes were found in both the viral tk and pol coding regions of HSV-1 N4, characterization of a recombinant virus expressing the HSV-1 N4 polymerase suggested that the TK and Pol together conferred the acyclovir-resistance phenotype.

Characterization of herpes simplex viruses selected in culture for resistance to penciclovir or acyclovir.

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 Acv(r) 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 Pcv(r) 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.

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 10(4) plaque-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.