The demand for antiviral susceptibility testing has risen in response to the increased awareness that drug resistance can result from the widespread use of antiviral therapy. GCV has been the drug of choice for treatment of systemic CMV disease. However, long-term therapy and suboptimal drug concentrations increase the risk of development of GCV resistance. The UL97 gene is by far the most frequent site of mutations conferring GCV resistance, and extensive work by a number of groups (2
) has failed to identify any nucleotide changes associated with GCV resistance outside codons 460, 520, and 590 to 607 (Fig. ). Not all of the mutations at these sites have been confirmed by marker transfer. For some of the unconfirmed mutations, a phenotypically sensitive isolate has been obtained prior to the isolation of a resistant isolate from the same patient. Sensitive and resistant isolates from the same patient, which differ only in a specific point mutation or deletion in association with GCV therapy, provide strong support for the role of that mutation in GCV resistance.
A genotypic assay designed to detect several of the UL97 mutations has been used by a number of investigators (2
). The assay is based on detection of loss or gain of restriction enzyme cleavage sites directly resulting from the presence of UL97 GCV resistance mutations. There are several advantages to this type of assay. The assay is rapid because it can be performed directly from the initial laboratory tube culture, it relies on PCR amplification of short sequences surrounding each potential mutation site, and it requires relatively simple equipment to perform. However, potential problems may be encountered if products are incompletely digested, new restriction sites are created by nucleotide changes unrelated to resistance, or resistance results from a DNA polymerase mutation. In addition, previously unmapped resistance mutations will not be detected.
We believe that automated sequencing represents the state-of-the-art approach to genotypic detection of drug resistance. The results of the sequencing of the UL97 gene from GCV-sensitive isolates demonstrate that the UL97 gene is highly conserved among clinical isolates, with an average 99% sequence identity. These baseline sequences along with the well-defined sites for drug resistance mutations provide the foundation for a rapid genotypic drug resistance assay based on direct sequencing of PCR-amplified products containing these sites.
The assay as outlined in this report has a minimal requirement of visible cytopathic effect in the original tube culture obtained from the clinical laboratory, which usually requires 7 to 14 days postinoculation. Amplification and sequencing can be completed within approximately 48 h. This approach drastically shortens the time to detection of drug resistance mutations compared to the plaque reduction assay, which requires at least 2 weeks of propagation to acquire enough virus. In addition a minimum of a week is required before the assay can be read. The potential exists to further shorten the time required for the genotypic assay by extracting viral DNA directly from patient samples. Portions of the UL97 and polymerase coding sequences can be amplified from DNA extracted from blood cells or plasma (35
; N. S. Lurain, unpublished data) and directly sequenced to detect resistance mutations. We are currently focusing on developing this modification of the assay.
The primers listed in Table for amplification and sequencing of the viral templates have been used successfully in the assays that we have performed. Some of the isolates contain single base changes within the sequencing primer binding sites, but there was no apparent effect on the ability of these primers to produce the predicted sequences. It is possible that future isolates may contain base changes that will affect either amplification or sequencing reactions. We have successfully tested several alternative primers (Table ) for both types of reactions; thus there is considerable flexibility in primer selection, although problems in primer design can arise from the high G+C content of the UL97 coding region.
By monitoring sequential isolates from the same patient over the course of long-term antiviral therapy, the appearance of known resistance mutations can be observed (2
). In many cases double peaks representing mixed sensitive and resistant viral populations become apparent on sequencing chromatograms as resistance develops. Another advantage of closely monitoring patients on GCV therapy is that new mutations potentially conferring resistance can be detected when sequences of drug-sensitive and drug-resistant strains from the same patient are compared.
Although this assay was developed specifically for GCV resistance resulting from UL97 mutations, the same approach can be used for genotyping drug-resistant DNA polymerase mutants. We have previously reported PCR conditions for amplification of the complete polymerase gene coding region. PCR products were sequenced to determine the variability of the DNA polymerase gene in a large group of drug-sensitive CMV isolates (10
). Detection of drug resistance mutations in the polymerase gene, however, is more difficult than detection of such mutations in the UL97 gene, because the polymerase gene is larger and the codons known to confer resistance are widely dispersed across the coding sequence (11
). Therefore, a genotypic assay for the polymerase gene will require a minimum of six sequencing reactions to cover the presently known sites of drug resistance mutations in the coding strand. Further sequencing and marker transfer experiments will also be required to determine whether there are as yet unidentified polymerase drug resistance mutations in phenotypically resistant CMV isolates.
Phenotypic assays are still important to corroborate genotypic results, because the genotypic approach assumes that the same drug resistance mutation in all genetic backgrounds will confer resistance. Phenotypic assays are also still required to identify targets of new drugs. While phenotypic assays have the advantage of relating drug resistance directly to the biological functions of the virus, the time required to perform the assays is too long to provide useful therapeutic information. The genotypic assay that we have developed for UL97 mutants is very rapid and covers all of the known drug resistance mutations. This method can be readily expanded and adapted to include the complete UL97 gene, the polymerase gene, and other viral targets to identify mutations conferring resistance to new antiviral drugs.