Infection and reinfection with multiple cytomegalovirus (CMV) strains have been shown to occur in immunocompromised individuals, sexually transmitted disease clinic attendees, and children attending day care centers. To characterize the CMV diversity in healthy seropositive individuals, 16 CMV PCR-positive specimens from 113 seropositive women were analyzed for glycoprotein gN and gB genotypes by cloning, followed by nucleotide sequencing of the plasmid DNA and/or restriction fragment length polymorphism (RFLP). The results showed that most (93.7%) of the PCR-positive specimens contained multiple gN and/or gB genomic variants, suggesting that the majority of women were infected with more than one virus strain. The results also showed that the RFLP technique might not be sufficiently sensitive to detect all of the genomic variants present in a sample.Cytomegalovirus (CMV) species are important opportunistic agents in infection of immunocompromised individuals and a frequent cause of congenital infection. Infection with multiple strains of CMV has been shown to occur frequently in immunocompromised individuals and sexually transmitted disease (STD) clinic attendees (8, 10). In addition, reinfection with different CMV strains was documented to occur in children attending day care centers (2). More recently, CMV reinfections were demonstrated in seropositive women, and such reinfections can result in intrauterine transmission and damaging fetal infection (5).Extensive genetic polymorphisms in envelope glycoproteins of CMV, including glycoprotein B (gpUL55), glycoprotein O (gpUL74), and glycoprotein N (gpUL73), have been demonstrated among clinical CMV isolates. Major envelope glycoprotein B (gB) of CMV has been demonstrated to elicit a strong neutralizing antibody response (6), and on the basis of restriction fragment length polymorphism (RFLP) analysis of clinical samples, four unique genomic variants, gB types 1 to 4, have been identified (9). Glycoprotein N has been shown to be highly polymorphic at the amino-terminal region, and most clinical CMV isolates have been shown to cluster into four distinct genomic variants, gN-1, gN-2, gN-3a, gN-3b, gN-4a, gN-4b, and gN-4c (11). Recent studies have shown that a significant proportion of the virus-neutralizing response was also directed against the gM/gN complex (17). No linkage between gN genotypes and gB genotypes has been observed (11).Published studies using RFLP analyses to determine the gN genotypes have identified only a single gN type in a given sample (11, 13). Studies of glycoprotein B based on RFLP analyses showed the presence of a single genotype or a limited number of samples containing mixtures of two gB genotypes (3, 7). However, a recent study using hybridization with typespecific probes (10) showed mixtures of all genotypes. To determine the CMV strain diversity in healthy seropositive women, the presence of multiple gN and gB genomic variants in urine or peripheral blood was examined by two different methods, RFLP and cloning followed by nucleotide se...