This is the first evaluation of the UL23 thymidine kinase gene of HSV-2, associated with ACV resistance, that focuses on specimens from the developing world, mostly Africa. Most of the subjects were receiving daily suppressive ACV therapy, for a mean duration of 12.1 months, at the time that HSV-2 DNA was recovered from a genital site. Slightly under half (40.6%) of the subjects had a known risk factor for ACV resistance, HIV infection, at the time of specimen collection (25
). We found no evidence of known genotypic mutations associated with decreased susceptibility to ACV among 68 specimens collected from 64 individuals during three phase III RCTs. Although we did not detect genotypic evidence of ACV resistance in any sample, we documented marked genetic variation that was higher than that observed in any other HSV locus.
The mainstays of anti-HSV therapy are ACV; its prodrug, valacyclovir; and the related drug famciclovir. ACV resistance is readily diagnosed by classical virologic methods, using graded concentrations of ACV and one of several cell culture-based readouts (23
). However, such testing for resistance is feasible only if a live viral isolate is available. For our study, only DNA specimens were obtained, due to storage and transport issues at geographically remote sites. This limited our ability to easily interpret the significance of the amino acid coding variants that we observed. One strength of analyzing DNA collected directly from subjects, however, is that potential changes in DNA sequences associated with passage in vitro
, as has been documented for some HSV loci (20
), are avoided.
Even with the use of direct swabs, it is still possible for sequence errors to result from PCR. The reported error rate for Taq
polymerase is on the order of 1 error per 10−4
nucleotides per duplication cycle (or less) (2
). With 35 cycles of amplification and an amplicon length of 1,128 bp, we estimated about 3.5 errors per amplicon. The proofreading function in specific thermostable polymerase products used in this project is reported to decrease the error rate by 6- to 8-fold, leading to an estimated 0.5 errors per final sequence. We believe that most of our newly reported variants are accurate for the following reasons. First, the newly reported sequence variants that we observed independently in more than one specimen (T90C, A660G, C783T, G207A, A307G, G449A, and C582T) (Table ) are likely to be real polymorphisms, because identical PCR errors are very unlikely to have arisen independently in separate reactions. Second, some of the variants observed only once, such as T621C, C811A, and C895T, were all found in the same specimen from Pucallpa in mid-Amazonia in Peru (strain 33 in Fig. ). It is very unlikely that PCR would introduce three separate changes into a single amplicon of this length. This is likely a unique, geographically isolated strain. Third, some of the UL23 variants we observed only once (G79A, T85G, C476C, and G643A) were also detected in a previous survey (6
) of Australian HSV-2 cultures that used a less stringent polymerase. While PCR artifacts occur nonrandomly, it is unlikely that the same exact errors would occur independently.
It is also possible that we missed some mutations associated with acyclovir resistance because of our focus on UL23. Changes in the HSV DNA polymerase gene can alter its inhibition by ACV triphosphate. These mutations make a minor contribution to clinical ACV resistance (1a
We did not detect any obvious resistance-associated frameshift mutations, but we did detect a large number of novel coding mutations. Coding point mutations associated with ACV resistance have also been described (6a
), but unlike in the example of human CMV and ganciclovir resistance (18
), obvious clustering of such resistance-associated point mutations in hot spots has not yet been recognized. We did detect two point mutations (L228I and R338Q) that map near known ACV resistance-associated sequence variants (D229H and C336Y, respectively) (12
). In addition, the H165D amino acid substitution, which carries a charge change from positive to negative, observed in a specimen collected in Zambia from an HIV-seronegative subject in the HPTN 039 study after 267 days of ACV therapy, is near the putative nucleoside binding domain of HSV-2 thymidine kinase at amino acids 169 to 177 (29
). Unfortunately, technical limitations precluded the storage or direct recovery of viable virus strains suitable for direct testing of ACV susceptibility. There are technologies available to determine the possible functional significance of the coding variations we have detected. They include the expression of recombinant protein and ACV phosphorylation assays in vitro
) or the creation of chimeric viruses containing the UL23 sequences from this study inserted into a thymidine kinase-deficient strain, followed by phenotypic resistance testing.
Studies from developed countries have documented in vitro
ACV resistance in approximately 0.28 to 0.33% of HSV isolates from immunocompetent individuals (5
). Interestingly, ACV use is not generally associated with drug resistance in immunocompetent persons at either the individual or population level (1a
). The frequency of resistance is typically higher in immunocompromised individuals, including those with HIV infection, where 4 to 7% of isolates in developed countries may be resistant to ACV (25
), although most source patients are also without clinical evidence of resistant disease. These strains are generally felt to arise due to selective pressure from ACV in the absence of a strong antiviral contribution from host immunity.
We did not detect sequence clusters that correlated with the geographic origins of our specimens. This contrasts with data from Norberg et al., who sequenced regions of the HSV-2 genes US7 and US8 from 20 isolates from Scandinavia and 27 isolates from Tanzania. Two straggling clades were detected, one of which contained only Tanzanian strains while the other included sequences from both regions (22
). In our data set, sequences from Peru were admixed with African sequences in all major branches of the UL23 neighbor-joining tree. Kaneko et al. sequenced 36 HSV-2 genes from 36 subjects in Japan (15
). They analyzed UL3, US1, and US4, their most divergent loci, as trees and noted that they had differing topologies. These data, and coanalysis of US4 data with the US7/US8 data set by Norberg et al., are consistent with crossovers and recombination in HSV-2 molecular evolution (22
The UL23 locus studied in this report seems particularly useful for strain tracking, with variants detected at 3.4% of nucleotides among 68 sequences. The most divergent strains, 31 (Pucallpa, Peru) and 57 (Mwanza, Tanzania), differed at 0.7% of the nucleotides. Several variable loci have rare alleles that are reasonably prevalent, so that single-nucleotide polymorphism (SNP) typing schemes could be useful. This variation is higher than the maximal 0.4% variance noted by Norberg et al. among 47 strains sequenced in the US4, US7, and US8 regions. Kaneko et al. noted that many HSV-2 genes were completely invariant in Japan (e.g., UL4, UL5, UL12, and 9 other genes) (15
). The low dN
ratio observed for UL23 is consistent with active selection to preserve certain amino acid sequences despite the apparent tolerance for amino acid changes in many locations.
The HIV-infected persons we studied had relatively preserved CD4 T-cell counts, in contrast to the advanced immune deficiency typically reported in HIV/HSV-2-coinfected persons who develop clinically ACV-resistant HSV-2 (9
). These findings are encouraging, given the recommendation by WHO to add ACV to the treatment regimen for genital ulcer disease (32
). Although the clinical trials from which our specimens came did not detect a decrease in HIV transmission, there were beneficial effects of ACV, such as decreased disease progression among HIV-infected persons being treated with ACV in the Partners in Prevention trial and a reduced frequency of genital ulcerations (19
). Recent biological findings that may explain the poor biological activity of ACV at the doses used in the recent trials include the observation of frequent shedding of HSV-2 in the genital tract despite administration of standard doses of antiherpesviral drugs (A. Wald, C. Johnston, M. Saracino, L. Olin, K. Mark, S. Selke, M. Huang, and L. Corey, presented at the 18th Meeting of the International Society for Sexually Transmitted Diseases Research, London, England, 28 June to 1 July 2009) and the persistence of HIV-susceptible, CCR5- or CXCR4-expressing, HSV-2-specific CD4 T cells in the genital skin at sites of previous HSV-2 lesions despite several months of antiherpesvirus therapy (33
). In addition, ACV suppression of genital shedding of HSV-2 was not as effective at South American and African sites as at North American sites within the HPTN 039 study, for unknown reasons (11
). Improved antiherpesvirus regimens might show activity against HIV transmission, and clinical trials should continue to monitor for the possible development of resistance to anti-HSV drugs in geographically distinct populations.