Phenotyping of Cytomegalovirus Drug Resistance Mutations by Using Recombinant Viruses Incorporating a Reporter Gene

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2005, p. 2710 2715 Vol. 49, No. 7 0066-4804/05/$08.00 0 doi:10.1128/aac.49.7.2710 2715.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved. Phenotyping of Cytomegalovirus Drug Resistance Mutations by Using Recombinant Viruses Incorporating a Reporter Gene Sunwen Chou,* Laura C. Van Wechel, Heather M. Lichy, and Gail I. Marousek Medical and Research Services, VA Medical Center and Oregon Health & Science University, Portland, Oregon Received 30 November 2004/Returned for modification 8 February 2005/Accepted 3 March 2005 A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC 50 ). Changes in the IC 50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate. Treatment of cytomegalovirus (CMV) infections with current antiviral drugs ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) may select for viral drug resistance mutations. CMV UL97 phosphotransferase gene mutations at codons 460, 520, and 590 to 607 have been shown to confer ganciclovir resistance (1, 4), while an increasingly diverse set of viral UL54 DNA polymerase (pol) mutations confer varying degrees of resistance and cross-resistance to the three drugs (2, 3). Resistance of CMV to antiviral drugs may be diagnosed genotypically, by looking for known viral resistance mutations in UL97 or pol, or phenotypically, by applying various drug concentrations to a growing virus in cell culture. Genotypic analysis is rapid and can be performed without the need for viral isolation, but mutations of indeterminate significance for resistance continue to be reported without phenotypic validation (8) and the degree of resistance conferred by combinations of mutations may be difficult to deduce by genotypic testing alone. Phenotypic analysis detects drug resistance without the need for genetic information, but even if a CMV isolate is available for testing, the standard plaque reduction assay is slow, technically demanding, and shows a high degree of interlaboratory variability (6). For diagnosis of CMV drug resistance in a clinical setting, genotypic methods are more practical, but this requires the continuing study of phenotypes corresponding to new mutations encountered in clinical isolates. The drug resistance phenotype corresponding to specific viral mutations can be determined by transferring them to a known drug-sensitive reference strain and testing for the resulting change in drug sensitivity. In the past few years this process, known as marker transfer or recombinant phenotyping, has been facilitated by the development of CMV strains * Corresponding author. Mailing address: VA Medical Center, P3ID 3710 SW U.S. Veterans Hospital Road, Portland, OR 97239. Phone: (503) 273-5115. Fax: (503) 273-5116. E-mail: chous@ohsu.edu. containing unique restriction sites in UL97 or pol that permit the rapid generation of recombinant viruses containing desired mutations (2, 4). Phenotyping of the resulting recombinant viruses then becomes the rate-limiting step, especially when performed by the standard plaque reduction assay. Here, we report the construction of CMV strains containing unique restriction sites in UL97 and pol, along with a secreted alkaline phosphatase (SEAP) reporter gene inserted at the nonessential gene region US6. This enabled the generation of recombinant viruses containing desired mutations in either or both UL97 and pol, which could be quantitated by simply measuring the SEAP activity in the culture supernatant. Reduction in SEAP activity under drug was used to determine drug sensitivity. Results of transferring specific mutations were compared with results previously obtained using traditional phenotyping assays, and the effects of combinations of mutations were studied for the first time. (Presented in part at the Infectious Diseases Society of America Annual Meeting, Boston, Mass., October 2004 [abstract 641] and the 44th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, October 2004 [abstract 615].) MATERIALS AND METHODS Viruses and plasmid transfer vectors. CMV strain AD169 (ATCC VR-538) was obtained from the American Type Culture Collection. All CMV strains and recombinants were propagated in human foreskin fibroblasts. Mutagenesis in the viral UL97 phosphotransferase and UL54 DNA polymerase (pol) genes was accomplished by homologous recombination using plasmid transfer vectors analogous to those previously published (2, 4). In the UL97 region, the starting plasmid was a Bluescript subclone of the AD169 genome (GenBank X17403) from nucleotides 139690 to 144436 (NotI and KpnI restriction sites, respectively). In the pol region, the corresponding subclone contained AD169 nucleotides 74828 to 81436 (XbaI and EcoRI restriction sites, respectively). Desired nucleotide changes (unique restriction sites PmeI or SwaI, or resistance-related mutations) were introduced into these transfer vectors by replacement of segments of these subclones by PCR products as described elsewhere (2, 4). A green fluorescent protein selectable marker (hrgfp) or SEAP reporter 2710

VOL. 49, 2005 CMV RECOMBINANT PHENOTYPING 2711 Recombinant strain Parental strain (genomic DNA) TABLE 1. Genotypes of AD169-derived recombinant CMV strains Genotype UL97 pol US6 region Selection marker T1421 AD169 wt a PmeI (453), PmeI (S897L) P522S V7811 wt GCVr FOSr T1937 T1421 wt PmeI (453), PmeI (S897L) wt None T2025 T1937 SwaI (H587Y) L595S PmeI (453), PmeI (S897L) wt GCVr T2065 T2025 SwaI (H587Y) PmeI (453), PmeI (S897L) MIE-hrGFP GFP T2211 T2065 SwaI (H587Y) PmeI (453), PmeI (S897L) MIE-SEAP None a wt, wild type. gene, both driven by CMV major immediate-early (MIE) promoters, were targeted for insertion at CMV US6 by flanking the expression cassettes with 2 kb of US3 sequence on one side and of US6 sequence on the other side, each incorporating PacI restriction sites at the junctions. This region of the CMV genome is dispensable for normal viral replication in cell culture. The hrgfp expression vector (MP234) was obtained from Mark Peeples (Rush Presbyterian St. Lukes Medical Center, Chicago, Ill.). The SEAP expression vector gwizseap was obtained from Gene Therapy Systems (San Diego, CA). Construction of reference strain T2211. Strain T2211, containing unique PmeI sites in pol, a SwaI site in UL97, and a SEAP expression cassette at US6, was constructed by successive recombination steps producing intermediate viral strains as shown in Table 1. Strain AD169 genomic DNA was extracted from infected fibroblast cultures as previously described (4). The AD169 sequence (GenBank X17403) contains no restriction sites for enzymes PacI, PmeI, or SwaI. To introduce PmeI restriction sites at codons 453 and 897 of the pol gene, a transfer vector containing PmeI sites at those codons and drug resistance markers P522S and V781I was prepared and cotransfected with strain AD169 DNA, followed by selection for recombinant virus T1421 using GCV and FOS, as described previously (2). The drug resistance markers were then removed from T1421 by another round of recombination (2). In the next round, a unique SwaI site was inserted at codons 584 to 586 of UL97 using the same approach as described elsewhere (4) of cotransfection with a GCV resistance marker, L595S, and its subsequent removal. Simultaneously, an hrgfp expression cassette was inserted at US6 by cotransfection and homologous recombination, and fluorescent cells infected with recombinant virus (T2065) were selected by fluorescenceactivated cell sorting. Finally, strain T2065 was plaque purified and its genomic DNA was extracted, digested with PacI, and cotransfected with the SEAP expression cassette targeted at the US6. The resulting nonfluorescent recombinant virus (T2211) (Table 1) was triply plaque purified, and its genotype was verified by PCR amplification and sequencing of the UL97, pol, and US3-US6 gene regions. Strain T2211 was then used as the reference virus for phenotyping and for construction of additional recombinant viruses containing mutations of interest (marker transfer). Marker transfer. Resistance-related mutations in CMV UL97 or pol were transferred into T2211 by cotransfection of T2211 DNA digested with SwaI or PmeI, respectively, and a transfer vector incorporating the desired mutation in the desired gene region. Recombinant virus that grew (usually in 10 to 14 days) was screened by PCR and restriction analysis or DNA sequencing for the desired mutation and was plaque purified thereafter, all without using any drug selection in cell culture. The plaque-purified recombinant viruses were sequenced in the entire coding sequence of the modified genes to confirm the presence of the desired mutations and absence of extraneous ones. Sequencing was performed by dye terminator chemistry (BigDye v1.1; Applied Biosystems) on the ABI377 automated sequencer. Phenotypic assays of recombinant viruses. Cell-free virus stock was prepared from each CMV strain and titrated for infectivity by enumeration of cells staining positive at 24 h for CMV MIE antigen (2). Growth curves were performed at a high and low multiplicities of infection (MOI) to compare the increase of viral infectivity of strains T2211 and AD169. Over 4- and 7-day periods, daily aliquots of culture supernatant were collected and stored at 80 C and the removed medium was replaced with fresh medium. The stored aliquots were then assayed for infectivity by inoculation on fibroblast monolayers and MIE antigen staining (2). SEAP assays were performed by diluting culture supernatant 1:10 (or 1:4 for samples collected the day after inoculation) in dilution buffer (50 mm Tris [ph 7.4], 150 mm NaCl) and heating to 65 C for 30 min. Thirty microliters of this diluted supernatant was then mixed with an equal volume of assay buffer (2 M diethanolamine, ph 9.8, and 28 mm L-homoarginine; both from Sigma), followed 5 min later by 30 l of ready-made chemiluminescent substrate mixture (0.4 mm CSPD with Emerald II enhancer in 0.1 M diethanolamine; Applied Biosystems Tropix). After 15 min, chemiluminescence (in relative light units [RLU]) was read in a 96-well plate luminometer (Glo-Runner; Turner Biosystems). All RLU data here are reported as the luminescence per 30 l of undiluted culture supernatant. Yield reduction assays for drug resistance were performed by inoculating 6 wells of a 24-well culture of human foreskin fibroblasts that were confluent for 3 days with 0.3 ml of virus to be tested, at an MOI of 0.01 to 0.03, corresponding to a SEAP RLU of 400 to 1,500 at 24 h (see Results). After 90 min, the inoculum was removed and the culture medium was then replaced in the wells, five of which contained twofold serial dilutions of drug (GCV, FOS, or CDV) above and below typical inhibitory concentrations (see Results). Four to 7 days after inoculation, aliquots of culture supernatant from wells with and without drug were assayed for SEAP activity as described above. The drug concentration (IC 50 ) required to reduce the SEAP activity to 50% of the control value (in the well without added drug) was calculated by fitting an exponential curve to the SEAP activities measured in the wells containing various drug concentrations. The IC 50 values of at least five separate experiments with each drug and virus were averaged, and results are reported as mean and standard deviation values. The ratio of IC 50 value to that of a sensitive control strain assayed at the same postinoculation interval was used as an index of drug sensitivity and was compared to values in the existing literature for the mutations being studied. RESULTS Properties of CMV strain T2211. Strain T2211 was derived from strain AD169 after several recombination steps, as listed in Table 1, resulting in the genotype shown. The insertions of unique restriction sites in UL97 and pol were accompanied by single amino acid changes at codons 587 and 897, respectively. T2211 also contained a SEAP gene cassette at US6 driven by a copy of the MIE promoter (Fig. 1). Despite these genetic changes, strain T2211 had the same cytopathic effect in culture, and similar infectivity growth curves at both high and low MOI, as the parental strain, AD169 (Fig. 2). Strain T2211 appeared to be slightly slower growing, but this did not affect the ability to prepare high-titer cell-free virus stock or obtain normal yields of genomic viral DNA for transfection. SEAP activity was readily detectable in culture supernatants of strain T2211 and its derivatives at 24 h postinoculation, and the activity as measured by chemiluminescence (in RLU) at this time point was directly related to the MOI (Fig. 3). After infection at an MOI of 0.01 to 0.03, accumulated SEAP activity in the culture supernatant increased to 30,000-fold over baseline at 6 days and reached a plateau shortly thereafter. At days 4 to 6, the rate of increase in SEAP activity was comparable to that of viral infectivity (Fig. 2). The growth kinetics of T2211 under different concentrations of GCV, FOS, and CDV were used to determine the optimum day of assay of SEAP activity for purposes of drug sensitivity phenotyping. Based on the shape of the growth curves (Fig. 4), days 5 or 6 postinoculation appeared to be the best times for determining IC 50

2712 CHOU ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 1. Genome map of recombinant CMV strain T2211. This strain was derived in several steps from strain AD169 (Table 1) and incorporates unique restriction sites in the viral pol and UL97 gene regions, as well as a SEAP reporter gene at US6 driven by the CMV major immediate-early promoter. values because virus (SEAP activity) was still growing exponentially both with and without drug. For reproducibility of IC 50 values, the MOI was kept between 0.01 and 0.03 as judged by the SEAP activity at 24 h (400 to 1,500 RLU) (Fig. 3). Marker transfer (recombinant phenotyping). New recombinant strains containing desired sequence changes in UL97 or pol were created by digestion of T2211 DNA followed by homologous recombination. Control strains were derived to show the phenotypic effects of amino acid changes in UL97 and pol resulting from insertion of the restriction sites in strain T2211. The wild-type amino acid sequences in UL97 and pol were restored in strains T2233 and T2241, respectively. The drug sensitivity phenotypes for all three drugs, GCV, FOS, and CDV, were not significantly different for strains T2211, T2233, and T2241 (Table 2), indicating a lack of phenotypic effect from the amino acid changes in UL97 and pol in strain T2211. The sensitive strains showed IC 50 values for GCV, FOS, and CDV (Table 2) that were comparable to those reported with plaque reduction, although the latter assay showed very wide interlaboratory variation (6). For example, published strain AD169 plaque reduction IC 50 values (6) include, for GCV, 1.0 to 9.6 M (mean, 4.0) and, for FOS, 35 to 112 M (mean, 62). Other phenotyping assays may give somewhat different results; the DNA hybridization method used for some of the historical phenotypes in Table 2 gave a lower AD169 GCV IC 50 value of 0.55 to 0.74 M (1). Based on the standard deviations of the IC 50 determinations (Table 2), mutations conferring twofold or greater changes in IC 50 values should be reliably identified. The most common UL97 GCV resistance mutations (1, 4) were transferred into T2211, creating the recombinant strains listed in Table 2. Yield reduction IC 50 values showed the resistance mutations M460V, A594V, and L595S to confer 7- to 10-fold increases in the IC 50 for GCV, similar to what had previously been reported for these mutations (1), and the mutation C592G to confer a more modest 2.5- to 3-fold resistance to GCV, as previously reported for a plaque reduction assay (4). The D605E change is the only reported sequence polymorphism found in baseline sensitive CMV isolates in the UL97 codon range 590 to 607, where GCV resistance mutations are FIG. 2. Growth curves of CMV strains AD169 and T2211. Cultures were inoculated at the indicated MOI, and the culture supernatants were sampled at intervals for the amount of infectious virus. Day zero values represent the infectivity of the input virus. In parallel, SEAP activity (RLU) in the supernatant was measured for T2211 cultured at an MOI of 0.03. Each data point and error bar represents the mean standard error of the mean of three experiments.

VOL. 49, 2005 CMV RECOMBINANT PHENOTYPING 2713 clustered (7). As expected, the transfer of this amino acid change (strain T2278) did not confer any change in GCV sensitivity (Table 2). It was reported that the D605E polymorphism reversed the GCV resistance conferred by the mutation A594P in an expressed enzyme assay system (5). To examine whether D605E affected the phenotype conferred by the four most common UL97 resistance mutations, we transferred D605E in combination with each of the mutations, L595S, A594V, M460V, and C592G. Results (Table 2) showed that the differences in IC 50 values for the mutations with and without D605E did not exceed the standard errors of the assays. A known pol resistance mutation (deletion of codon 981-2 or del981-2) was chosen for transfer into T2211 because it confers resistance to all three drugs, GCV, FOS, and CDV (3). This resulted in strain T2222. As shown in Table 2, the triple resistance phenotype as measured by SEAP IC 50 values for the pol del981-2 mutation was quantitatively similar to that previously determined by plaque reduction (3). To demonstrate the effect of combined UL97 and pol mutations, a double mutant strain T2261 was derived from T2222 by digesting its genomic DNA with SwaI and recombination with a transfer vector containing UL97 C592G. The double mutant containing UL97 mutation C592G and pol mutation del981-2 showed much higher GCV resistance (Table 2) than strains containing either mutation alone (T2222 or T2258). DISCUSSION FIG. 3. Multiplicity of infection versus SEAP activity at 24 h. Cell cultures were inoculated at various multiplicities of infection up to 0.04, and the SEAP activity in culture supernatants was assayed at 24 h postinoculation. Uninoculated cell culture supernatants had a background RLU reading of 25. The data points were fit to a logarithmic curve, giving the correlation coefficient (R 2 ) shown. Construction of a reference CMV laboratory strain T2211 containing unique restriction sites, together with a SEAP reporter gene, enabled the efficient recombinant phenotyping of mutations in the CMV UL97 and pol genes associated with drug resistance. This technical approach was validated by transfer of the most common UL97 GCV resistance mutations and a pol mutation conferring multidrug resistance. Extending previous work, the synergistic effect of combined UL97 and pol mutations on GCV resistance is now proven by marker transfer, and the status of UL97 D605E as a sequence polymorphism without effect on GCV resistance has also been demonstrated. For the known resistance mutations transferred into strain T2211, the amounts (ratios) by which the SEAP yield reduction IC 50 values increased over a sensitive control strain are closely comparable to values previously reported for the same mutations. The absolute IC 50 values vary because of differences across assays and laboratories, but most of the common UL97 mutations observed in clinical isolates have been reported to confer a 5- to 10-fold increase in GCV resistance, while some others confer only a borderline 2- to 3-fold increase (4). With the current SEAP-based phenotyping system, it is confirmed that the UL97 mutation C592G confers a lesser degree of GCV resistance than M460V, A594V, and L595S and that the pol mutation del981-2 confers multidrug resistance. Although the UL97 C592G mutation and the pol del981-2 mutation individually confer only modest GCV resistance, the combination of the two confers a much higher level of resistance, as previously hypothesized but not proven by marker transfer (9). FIG. 4. SEAP growth curves of CMV strain T2211 under GCV, FOS, and CDV. Cell cultures were inoculated at an MO1 of 0.01 and grown under various drug concentrations (A GCV, B FOS, and C CDV). SEAP activity in culture supernatants was assayed daily at 4 to 7 days postinoculation. Each data point and error bar is the mean standard error of the mean of eight experiments.

2714 CHOU ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Genotypes and phenotypes of T2211-derived CMV strains CMV strain Genotype a IC 50 (ratio) c by SEAP assay b Published marker transfer d (ratio) c UL97 pol GCV FOS CDV GCV FOS CDV Controls T2211 SwaI (H587Y) PmeI (S897L) 1.1 0.4 46 13 0.23 0.05 T2233 wt e PmeI (S897L) 1.2 0.4 51 3 0.25 0.07 T2241 SwaI (H587Y) wt 1.3 0.2 35 7 0.23 0.04 T2278 D605E PmeI (S897L) 0.9 0.3 Mutants T2259 M460V PmeI (S897L) 9.8 2.4 (8.3) 5.2 (7.0) T2256 M460V D605E PmeI (S897L) 11.5 3.1 (9.8) T2258 C592G PmeI (S897L) 3.5 1.1 (2.9) 45 5 (0.9) 0.31 0.07 (1.2) 14.5 (2.6) T2247 C592G D605E PmeI (S897L) 3.1 0.8 (2.6) T2255 A594V PmeI (S897L) 9.8 3.0 (8.3) 5.9 (10.7) T2235 A594V D605E PmeI (S897L) 9.1 3.7 (7.7) T2260 L595S PmeI (S897L) 10.9 3.4 (9.2) 6.4 (11.6) T2234 L595S D605E PmeI (S897L) 10.0 1.3 (8.5) T2222 SwaI (H587Y) del(981 982) 6.3 2.4 (4.8) 124 11 (3.5) 0.94 0.16 (4.1) 47 (6.1) 268 (4.0) 1.8 (5.1) T2261 C592G del(981 982) 22.2 8.2 (16.9) 144 19 (4.1) 1.31 0.26 (5.7) a Resistance mutations are shown in bold typeface. b IC 50 values are in M, expressed as mean standard deviation of at least five assays. c Ratio of IC 50 to that of sensitive control strain. d Previously published IC 50 and ratio for the same resistance mutations (1, 3, 4). e wt, wild type. The known UL97 GCV resistance mutations are tightly clustered at codons 460, 520, and the range of 590 to 607 (4). Most of the assorted amino acid changes (including deletions) that have been observed in CMV isolates at codons 590 to 607 are associated with varying degrees of GCV resistance (4), with the notable exception of D605E (7). Based on studies of recombinant vaccinia viruses expressing UL97, the D605E change was proposed to reverse the GCV resistance conferred by the unusual mutation A594P (5). Here, after examining the phenotypes of recombinant viruses containing the most common UL97 mutations with and without D605E, we confirm that D605E does not confer any GCV resistance, but we cannot confirm that the polymorphism has a significant effect on the degree of resistance conferred by the most common UL97 resistance mutations. It is possible that the effect of D605E is too small to measure in our assay system or that it occurs with only certain less-common UL97 mutations. The major advantages of this recombinant phenotyping approach are its relative rapidity and reproducibility, along with the ability to study combinations of UL97 and pol mutations. The entire process (several weeks) can actually be faster than propagating, quantitating, and testing a clinical isolate by conventional plaque reduction. Compared with clinical isolates, the high-titer extracellular virus produced by the laboratory strains and recombinants simplifies quantitative work. Because the SEAP reporter signal is measured directly from culture supernatants with minimal processing, fewer variables are introduced during the quantitation step. Use of multichannel equipment and a 96-well plate luminometer greatly increases throughput. These features permit the proper control of assay variables, such as the viral inoculum or culture duration, and allow the generation of sufficient replicate assays to assess the reproducibility of the results. Cultures may be nondestructively sampled at several time points to assess growth characteristics and optimize the timing of the assay. The various CMV phenotyping assays in current use measure different aspects of virus growth, and comparability of results must be empirically determined. Readouts of viral growth have been based on plaque formation, extracted viral DNA, extracted viral antigen, or antigen detection in infected cells (including flow cytometry). The current assay measures the activity of the CMV MIE promoter that drives the SEAP reporter gene. This promoter is most active early in the viral replication cycle, and the exponential increase in SEAP activity during multiple cycles of growth reflects the infection of new cells as well as the stability of the secreted enzyme. So far, we have validated this multicycle SEAP-based assay only for the current CMV drugs (GCV, FOS, and CDV), which all target the CMV DNA polymerase. Extension of this system to experimental CMV drugs with different mechanisms of action will require further study. Use of strain T2211 as a base for transfer of resistance mutations will be useful in studying new mutations, combinations of mutations, and clinical CMV sequences without an available viral isolate. With the adoption of molecular diagnostic methods for CMV, it is now common for the virus to be detected only by direct PCR amplification from clinical specimens, without an accompanying viral isolate. Previously uncharacterized sequence changes (both in UL97 and pol) associated with CMV infection poorly responsive to drug therapy (8) can be assessed more efficiently using this technical approach. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI-39938 and by Department of Veterans Affairs research funds. REFERENCES 1. Chou, S., A. Erice, M. C. Jordan, G. M. Vercellotti, K. R. Michels, C. L. Talarico, S. C. Stanat, and K. K. Biron. 1995. 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