Human cytomegalovirus haplotype reconstruction reveals high diversity due to superinfection and evidence of within-host recombination

Significance

Human cytomegalovirus (HCMV) is a major global cause of congenital disability and transplant-related morbidity. Excessive levels of within-host HCMV nucleotide diversity are attributed to unexpectedly high mutation rates. Here, we show that high HCMV diversity is due to the frequent presence of mixed infections with genetically distinct strains, whereas HCMV in nonmixed infections is no more diverse than other DNA viruses. Using serial patient samples, we reconstruct viral strain haplotypes to pinpoint the timing of HCMV superinfections occurring within the study sampling time frame and uncover within-host viral recombination. From these results, we identify likely sources of infection and demonstrate probable selection for recombinant viruses. These results generate new, yet testable, insights into putative viral and host drivers of HCMV evolution and pathogenesis.

Abstract

Recent sequencing efforts have led to estimates of human cytomegalovirus (HCMV) genome-wide intrahost diversity that rival those of persistent RNA viruses [Renzette N, Bhattacharjee B, Jensen JD, Gibson L, Kowalik TF (2011) PLoS Pathog 7:e1001344]. Here, we deep sequence HCMV genomes recovered from single and longitudinally collected blood samples from immunocompromised children to show that the observations of high within-host HCMV nucleotide diversity are explained by the frequent occurrence of mixed infections caused by genetically distant strains. To confirm this finding, we reconstructed within-host viral haplotypes from short-read sequence data. We verify that within-host HCMV nucleotide diversity in unmixed infections is no greater than that of other DNA viruses analyzed by the same sequencing and bioinformatic methods and considerably less than that of human immunodeficiency and hepatitis C viruses. By resolving individual viral haplotypes within patients, we reconstruct the timing, likely origins, and natural history of superinfecting strains. We uncover evidence for within-host recombination between genetically distinct HCMV strains, observing the loss of the parental virus containing the nonrecombinant fragment. The data suggest selection for strains containing the recombinant fragment, generating testable hypotheses about HCMV evolution and pathogenesis. These results highlight that high HCMV diversity present in some samples is caused by coinfection with multiple distinct strains and provide reassurance that within the host diversity for single-strain HCMV infections is no greater than for other herpesviruses.