This report demonstrates that there is great diversity among the strains that cause congenital CMV infections and congenitally infected neonates often harbor multiple CMV genotypic variants. Among the 28 saliva samples from infants obtained at birth and saliva, urine, and blood samples from 13 of the 28 subjects obtained at follow-up, all gN, gB and gH genotypes were found to cause congenital infection. Furthermore, mixed infection with >1 virus strain was detected in approximately one-third of the study infants. In addition, the presence of distinct virus strains in specimens from different compartments from the same child was demonstrated.
In studies of both immunocompetent and immunocompromised hosts, multiple CMV genotypes have been detected in older children and adults. Although studies have documented that these viruses are acquired over time through reinfections [4
], it is also possible that multiple viruses can be acquired at the time of primary CMV infection. It is generally believed that vertical transmission to the fetus occurs after maternal viremia. Whether this transmission occurs as a single event during pregnancy or through multiple placental transmission events over the course of the pregnancy is unknown. Studies of the guinea pig model of congenital CMV infection have shown that maternal viremia leads to placental infection, and, in some instances, a secondary viremia can occur and result in secondary seeding of the placenta. However, dissemination to the fetus is not always immediate and the placenta serves as a reservoir for the virus [15
]. Studies of human placentas have also shown multiple CMV genotypes at the maternal-fetal interface [18
]. Thus, virus transmission to the fetus resulting in congenital infection with multiple CMV strains could occur as a single infection with codisseminating strains, or by multiple transmission events of individual viruses, or both.
Studies examining the association between virus diversity at a single polymorphic gene and outcome in children with congenital CMV infection reported conflicting results [19
]. However, studies in the murine model and in immunocompromised patients suggest that coinfection with multiple strains of CMV could lead to enhanced pathogenicity [5
]. In a study that examined the diversity of 3 polymorphic CMV genes in infants with congenital CMV infection, >1 virus strain was detected in 8/10 specimens obtained from stillborn infants but only a single genotype from 22 living newborns [25
]. However, the interpretation of these findings is difficult because tissue-cultured viruses from living newborns were compared with paraffin-embedded tissue specimens from stillborn infants. Since propagation of virus in tissue culture often selects for a single virus strain [26
], the inability to detect multiple virus strains in infants with congenital CMV infection in previous studies [22
] could be due to methodological issues.
In the current study, original saliva, urine, and DBS samples from congenitally infected infants were analyzed to avoid tissue culture selection of viruses, and real-time PCR and cloning of PCR products were used to genotype virus strains. Previous studies examining genotype distribution have used either virus from tissue cultures or samples from urine only. In addition, genotyping was performed by gene amplification and sequencing of the product [22
]. Thus, it is likely that only dominant or selected genotypes were detected. Although the implications of finding that congenital infection can be caused by multiple CMV strains, with respect to sequelae such as SNHL, are not yet known, the results from the ongoing NIDCD CHIMES study [8
] that includes prospective follow-up of a large number of congenitally infected infants should provide a better understanding of the role of mixed infection on outcome.
In 13 study infants, urine, saliva, and blood samples were available within the first 3 weeks of life and were examined to determine the CMV strain diversity in different compartments. Although there was not a predominant genotype, unique strains in different compartments were found in 5 of these infants. Compartmentalization of CMV strains has been reported in immunocompromised populations [30
]. CMV has the ability to grow in certain cell types, and variable cell tropism is conferred by particular viral genes that are present in clinical CMV strains [32
]. This observation has raised the possibility that CMV strain variation might explain differences in the biological behavior of different virus strains. The finding that infants with congenital CMV infection can harbor multiple CMV genotypes, and that unique genotypes are found in different compartments, underscores the need for examining the relationship between strain variation and biological characteristics of viruses.
In 2 study infants, genotypes that were detected in saliva at follow-up (at 2–3 weeks of life) were different from those detected in the saliva samples obtained at birth. This finding could have multiple explanations. Although it is possible that these infants acquired new strains during the time between screening and follow-up, this is unlikely because the follow-up samples were obtained between 2 and 3 weeks of age. Alternatively, both strains may have been present at both time points but not detected in the screening samples because relatively low numbers of the minor virus populations were present. In a recent study, investigators examined plasma and bronchoalveolar samples from 9 immunocompromised patients using a highly sensitive deep sequencing method. All 9 patients had mixed infections with 1 or 2 dominant genotypes and several low-abundance genotypes. In addition, the prevalence of the individual genotypes was shown to change over time, with strains that were initially minor becoming dominant [6
]. The appearance of a “new” genotype not detected in the initial sample in our study infants may reflect a similar change in the prevalence of the individual strains.
A limitation of this study is that only a small proportion of the infected children identified in the NIDCD CHIMES study were included, which may have led to selection bias. However, this potential selection bias was unlikely to have affected the findings since the 28 study subjects have similar demographic characteristics as all CMV positive infants during the described time period. An additional limitation of the study is the use of real time PCR to detect gB and gH genotypes. The sensitivity of this assay is dependent on the relative amounts of viral DNA; thus, minor viral populations with low abundance of type-specific viral DNA could have been missed. This reduced sensitivity of the real-time PCR may have resulted in an overall underestimation of the true viral diversity within a sample and subject. Our conclusion that infection with multiple virus strains can occur in infants with congenital CMV infection remains valid, however. CMV is a large virus with >140 genes. In this study, only 3 loci (gB, gH, and gN, all known targets of neutralizing antibody) were examined to determine genetic diversity. Since many more polymorphic CMV genes have been identified, it is likely that the true virus strain diversity in the study population was underestimated. Although the relative frequency of mixed infection might change if a larger proportion of infected infants were included in the study and if more CMV loci were examined for diversity, the finding that some congenitally infected infants harbor multiple virus strains remains an interesting observation.
In summary, the present report demonstrates that there is great diversity in the CMV strains that cause congenital infection and that infection with multiple CMV strains occurs in congenital CMV infection. However, the relationship of specific genotypes and the implications of infection with multiple viral strains for the pathogenesis and long-term outcome in children with congenital CMV infection are not yet known.