Human Cytomegalovirus Genome Diversity in Longitudinally Collected Breast Milk Samples

Reactivation and shedding of human cytomegalovirus (HCMV) in breast milk during lactation is highly frequent in HCMV-seropositive mothers. This represents a key transmission route for postnatal HCMV infection, and can lead to severe disease in preterm neonates. Little is known about HCMV strain composition or intrahost viral population dynamics in breast milk from immunocompetent adults. We performed HCMV-specific target enrichment and high-throughput sequencing of 38 breast milk samples obtained in Germany between days 10 and 60 after giving birth from 15 mothers with HCMV DNA-lactia, and assembled HCMV consensus sequences de novo. The genotype distribution and number of HCMV strains present in each sample were determined by quantifying genotype-specific sequence motifs in 12 hypervariable viral genes, revealing a wide range of genotypes (82/109) for these genes in the cohort and unique, longitudinally stable strain compositions in each mother. Reactivation of up to three distinct HCMV strains was detected in 8/15 of mothers, with a statistical tendency for multiple-strain reactivations in mothers who had stated their country of origin as being outside Germany. As described previously, nucleotide diversity of samples with multiple strains was much higher than that of samples with single strains. Breast milk as a main source of postnatal transmission may serve as a repository for viral diversity and thus play an essential role in the natural epidemiology of HCMV.