INTRODUCTION

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Human cytomegalovirus (HCMV), a betaherpesvirus, has a large, complex genome consisting of approximately 240,000 nucleotide base pairs (bp) and over 200 open reading frames (20). Wild-type and laboratory strains of HCMV display nucleotide polymorphisms in several regions of the genome, especially in the a sequence, major immediate early (MIE), glycoprotein B (gB; UL55), and UL144 gene regions (7, 8, 12, 16-18; J. F. Bale, S. J. Petheram, M. Robertson, J. R. Murph, and G. Demmler, submitted for publication). Although the effects of these polymorphisms on the biology of HCMV infections are largely unknown, molecular variations can be analyzed to study the epidemiology and pathogenesis of HCMV infections (4, 5, 16).

Mapping the molecular profiles of HCMV has enabled investigators to determine the source and transmission patterns of HCMV infections. Studies of HCMV epidemiology have mapped strains by using analysis of the restriction fragment length polymorphisms (RFLPs) of the entire HCMV genome (1, 10), PCR-based analysis of a sequence variations (2, 22), DNA sequence analysis of polymorphic regions (11, 19; Bale et al., submitted), and stepwise analysis of the RFLPs of gene regions by using PCR-based methods (3, 7, 15, 18). Our laboratory has previously used a PCR-based algorithm that compares the size and RFLPs of the a sequence amplicon, the gB genotype as described by Chou and Dennsion (8), and the RFLPs of an amplicon derived by amplification of the MIE gene region (18). Although these molecular approaches can characterize HCMV strains accurately, they are time-consuming and available only in research laboratories.

Recently, Davis and colleagues reported that the HCMV genome contains numerous short tandem repeats (STRs) (9). The microsatellite regions consist of iterated motifs of one to six bases that occur in the genomes of eukaryotes and some prokaryotes and represent potential sites of mutation (14, 21). Davis and colleagues found at least 24 regions in the HCMV genome that exhibit length or sequence polymorphisms and suggested that analysis of certain polymorphic regions could enable laboratories to compare HCMV strains (9).

In this report, we confirm that analysis of STR length polymorphisms can be used to characterize HCMV strains. Forty-six HCMV strains, comprising 44 wild-type strains and 2 laboratory strains, Towne and AD169, were compared by PCR-based STR analysis using primers for 10 microsatellite regions. In each instance, STR analysis was concordant with the results of conventional analyses of strains based on DNA sequences and RFLPs. These results indicate that STR analysis provides a novel, effective means to compare the molecular profiles of HCMV strains.

	MATERIALS AND METHODS

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Virus strains. Forty-six HCMV strains, consisting of 2 laboratory strains, Towne and AD169, and 44 wild-type strains, isolated from 28 children and the mother of one congenitally infected child, were analyzed. Wild-type strains were collected in Iowa and Texas between 1986 and 1995 and were isolated on human foreskin fibroblast cells grown in shell vials or 24-well plates using methods described previously (3, 4, 16). Collection of HCMV strains was approved by the institutional review boards of the respective institutions, the University of Iowa and Baylor College of Medicine.

PCR primers. Twelve STR markers (Table 1; Fig. 1) were chosen from the group of 24 locations known to be polymorphic in HCMV (9). The primers were selected because they appeared to identify the most informative length polymorphisms and could be multiplexed into a single gel lane.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Linear map of the HCMV genome showing the relative sites of the STR loci analyzed in this study; adapted from data of Davis et al. (9).

PCR amplication and fragment analysis. Each locus was amplified in a 20-µl PCR mixture using 0.5 µl of unquantified target HCMV DNA. Reaction mixtures included 200 µM concentrations of each deoxynucleoside triphosphate, 10 mM Tris-HCl (pH 8.3), 40 mM NaCl, 1.5 mM MgCl₂, 0.2 mM spermidine-HCl, 0.25 U of Taq Platinum (Life Technologies, Gaithersburg, Md.), and 0.5 µM concentrations of each primer. Reaction mixtures for UL50C, UL38N, and UL95C also included 5% dimethyl sulfoxide.

Conventional genotyping and sequence analysis. HCMV strains studied by STR were analyzed by PCR-based methods and primers for the a sequence, gB, MIE, and UL144 HCMV gene regions using methods described previously (3, 15, 18). Strains were considered distinct if they displayed differences in the a sequence amplicon size, gB genotype, or MIE RFLP, or had <95% homology by UL144 or a-sequence nucleotide DNA sequences. Conversely, strains were considered indistinguishable if they displayed identical patterns for a sequence amplicon size, gB genotype, and MIE RFLP or had identical gB genotypes and 95% nucleotide sequence homology for a sequence and UL144 gene regions.

Analysis of STR results and comparison with conventional molecular strain analysis. Strains were coded, and STR analysis was conducted by persons unaware of the relationships of HCMV strains. Upon completion of STR analysis, strains were identified as distinct or indistinguishable by the STR method, and these results were compared with the results of conventional molecular analysis and the epidemiologic information regarding each strain.

	RESULTS

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STR polymorphisms. Of the 12 regions identified as showing potential length polymorphisms for the STRs of HCMV, 10 yielded results that were useful for strain comparison (Table 2). One region (UL46C) showed no polymorphisms among the 46 strains analyzed, and an additional region (UL38N) proved difficult to amplify and exhibited anomalous fragment mobility. The methods used in this analysis had high sensitivity, enabling detection of 1 to 3 bp differences between HCMV strains.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Stacked electropherograms showing the size polymorphisms among HCMV strains for three of the polymorphic STR regions (UL95C, UL50C, and UL23C), illustrating the data used to generate an HCMV STR map.

Comparison of HCMV strains. All strains yielded sufficient PCR product to allow comparison of STR patterns. Three strains failed to amplify for one region each, but the data for the remaining regions were adequate to allow valid characterization of each of these strains. Distinct STR patterns typically differed between strains at 5 to 7 sites of potential polymorphism. The AD169 and Towne strains showed distinct STR patterns and differed from each other as well as each of the wild-type strains. AD169 and isolate TX02 displayed the closest patterns of any distinct isolates, differing in 3 of 10 polymorphic regions.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   STR polymorphisms for three strains (TX25, 20009, and 501). Bands represent the size polymorphism for each of the STR loci, as described in Materials and Methods. The numbers 140 to 460 indicate the base pair size of PCR amplicons. Strains TX25 and 20009 display identical length polymorphisms for all loci. By contrast, strain 501 differs from the above strains at several loci (arrowheads).

Detection of simultaneous excretion of HCMV strains. Prior conventional analysis of strains 191A, 191B, and 191C yielded unusual results, suggesting genetic recombination or simultaneous shedding of multiple HCMV strains (3). In the present study these strains yielded STR patterns that suggested simultaneous excretion of two strains. Strains 191A and 191B each showed two STR polymorphisms at one or more loci, including regions UL122N, UL123I1N, and UL95C (Table 7). The most plausible explanation for the appearance of two alleles at each of these loci is the presence of two genetic variants, i.e., simultaneous shedding of two strains.

	DISCUSSION

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This report demonstrates that STR analysis accurately characterizes HCMV strain patterns. PCR analysis using primers for 10 microsatellite regions in the HCMV genome correctly identified HCMV strains shown to be distinct or indistinguishable by conventional molecular analysis. When allelic patterns were compared, distinct HCMV strains showed differences at three or more STR loci. With only one exception, indistinguishable strains had identical allelic patterns at all loci. The sensitivity of this method detects differences as small as 1 bp in STR patterns for each gene region. By analyzing multiple regions throughout the genome, STR analysis allows comprehensive comparisons of HCMV strains.

STR analysis has distinct advantages over prior molecular approaches to HCMV strain characterization. Current strategies, such as the one routinely used in our laboratory (18), require stepwise analysis of several gene regions for definitive comparisons of HCMV strains. Even though certain regions, e.g., the a sequence and UL144, are hypervariable, these regions have dominant genotypes, indicating that identical patterns can occur by chance if limited numbers of HCMV gene regions are examined. In contrast, multiplexed STR analysis allows simultaneous evaluation of several polymorphic regions, thus increasing efficiency of the analysis and reducing substantially the probability of chance associations.

STR also appears to facilitate detection of simultaneous excretion of distinct HCMV strains. Prior methods, such as genotyping of strains by analyzing the polymorphisms observed at the gB cleavage site (8, 13), have enabled investigators to identify mixed genotypes, which suggest excretion of two strains. Detecting multiple alleles at STR loci, however, provides convincing evidence of genetic allelism, a phenomenon possible only when two or more genetic variants, i.e., distinct HCMV strains, are present in a single sample.

A remarkable finding of this study was the observation that several Texas strains appeared to be identical to strains from Iowa City, Iowa. This observation was confirmed by nucleotide sequencing of a sequence and UL144 regions and by RFLP analysis of gB (Bale et al., submitted). We are unaware of any point source acquisition for these strains or any direct contact between the Iowa children and the parents of the Texas infants. This observation implies that the identical HCMV strains had a common ancestral origin. This conclusion requires that the STR patterns do not mutate during HCMV excretion and after transmission between individuals.

Although the STRs of eukaryotes have relatively high rates of mutation (20), the present data suggest that the HCMV STRs remain reasonably stable. This conclusion is supported by the identification of identical STR polymorphisms in (i) strains isolated 16 months apart from the same individual, (ii) strains isolated from children who had epidemiologic evidence for horizontal transmission (e.g., the isolates from the Cedar Rapids child care center), and (iii) a vertically transmitted strain from a mother-infant pair.

We selected the 12 primers according to the number of predicted size polymorphisms and the size of the predicted amplicons (9). By carefully modifying primers and selecting fluorescent tags, we were able to create an efficient multiplex of amplicons separated by size and fluorescent tags. One of the selected regions (UL46C) yielded no polymorphisms, and an additional region gave ambiguous results. These regions were dropped from the final analysis without affecting the ability to discriminate HCMV strains.

Because the amplified loci include mononucleotide repeats as well as di- and trinucleotide repeats, it was important to be able to repeatably score single base pair differences in amplicons exceeding 300 bp. To ensure crisp peaks and avoid ambiguity in markers with mono- or dinucleotide repeat polymorphisms, the reverse primers were synthesized with a 6-bp tail known to promote nontemplated nucleotide addition by Taq DNA polymerase (6, 12).

To promote accuracy and repeatability in DNA fragment sizing, we used an internal size standard with a 20-bp ladder. We had previously determined that a 10-bp size standard does not improve the accuracy of size determination or reproducibility compared with the 20-bp ladder (data not shown). Since the repeat regions were typically short (3 to 11 repeats), we observed few or no stutter bands of descending size, as can be seen in polymorphic repeat regions of greater length.

From a time and cost-management perspective, STR analysis is very efficient. The multiplex of 10 amplicons pooled for electrophoresis results in a turnaround time of less than 24 h, including PCR, gel electrophoresis, and scoring of alleles. The assay requires a specialized fluorescence detection platform, either slab gel or capillary, which is increasingly available in resource facilities providing DNA sequencing or DNA fragment analysis services. Although there is a relatively high initial cost in purchasing fluorescent-tagged primers (approximately $100 per pair), the amortized cost per assay is low since a single 100-nmol synthesis should be sufficient for approximately 8,000 assays. The Genomics Facility of the University of Utah calculates its costs at $1.20/genotype or $12.00 per HCMV strain.

In summary, these results confirm that wild-type HCMV strains possess STRs and that analysis of STR size polymorphisms accurately characterizes HCMV strains. The STR length polymorphisms appear to remain stable during prolonged excretion by an individual or after transmission to other individuals, a finding that has important implications for studies of the transmission and evolution of HCMV strains. STR analysis also allows identification of simultaneous excretion of multiple HCMV strains. Consequently, PCR-based analysis of STR size polymorphisms provides a robust method for comparing HCMV strains.