Deep Sequencing Reveals Highly Complex Dynamics of Human Cytomegalovirus Genotypes in Transplant Patients over Time

Abstract

In lung transplant patients undergoing immunosuppression, more than one human cytomegalovirus (HCMV) genotype may emerge during follow-up, and this could be critical for the outcome of HCMV infection. Up to now, many cases of infection with multiple HCMV genotypes were probably overlooked due to the limitations of the current genotyping approaches. We have now analyzed mixed-genotype infections in 17 clinical samples from 9 lung transplant patients using the highly sensitive ultradeep-pyrosequencing (UDPS) technology. UDPS genotyping was performed at three variable HCMV genes, coding for glycoprotein N (gN), glycoprotein O (gO), and UL139. Simultaneous analysis of a mean of 10,430 sequence reads per amplicon allowed the relative amounts of distinct genotypes in the samples to be determined down to 0.1% to 1% abundance. Complex mixtures of up to six different HCMV genotypes per sample were observed. In all samples, no more than two major genotypes accounted for at least 88% of the HCMV DNA load, and these were often accompanied by up to four low-abundance genotypes at frequencies of 0.1% to 8.6%. No evidence for the emergence of new genotypes or sequence changes over time was observed. However, analysis of different samples withdrawn from the same patients at different time points revealed that the relative levels of replication of the individual HCMV genotypes changed within a mixed-genotype population upon reemergence of the virus. Our data show for the first time that, similar to what has been hypothesized for the murine model, HCMV reactivation in humans seems to occur stochastically.Human cytomegalovirus (HCMV) is the most important viral pathogen affecting patients after solid organ transplantation (12,18,29). Lung transplant recipients have an especially high risk of acquiring severe and sometimes fatal HCMV infections, which are also associated directly or indirectly with graft rejection and bronchiolitis obliterans syndrome (6, 53).Human cytomegalovirus is a double-stranded DNA virus with a genome size of about 236 kb that is predicted to contain 165 protein-coding genes (9). Although most of the HCMV genome is highly conserved among the various HCMV strains, a subset of genes exhibits a high degree of variability, as has been shown by genome-wide sequence analysis as well as by examination of individual genes. A high level of genetic heterogeneity usually occurs in a limited number of distinct genotypes (9, 32, 33). Among the most variable genes are the viral envelope glycoprotein N (gN) and gO genes and the gene encoding the predicted membrane glycoprotein UL139. Sequence analysis of highly polymorphic regions within these three genes allows the discrimination of seven distinct gN genotypes (30, 31) and eight distinct gO and UL139 genotypes (3,24,35,45). The existence of such a large number of distinct genotypes provides a useful tool for investigating HCMV population diversity within a host.Reinfection with different HCMV strains is possible in the human host, and several strains can accumulate during...

Limits and patterns of cytomegalovirus genomic diversity in humans

“…The annualized rate of reinfection in healthy adults is as high as 10% (70), and multiple strain infections in transplant recipients also seem to be common (17). In this study, we analyzed the proportion of reinfections or mixed infections by studying the viral population divergence across time or compartments, estimating that approximately one-third of infections were mixed or reinfections.…”

“…The viral interhost diversity was first described through restriction fragment length polymorphism analysis (12) and was subsequently confirmed with targeted gene and genome-wide sequencing (7,8,13). In a similar manner, HCMV intrahost diversity has been described using genetic and genomic sequencing (10,11,(14)(15)(16)(17) and has been shown to be comparable with diversity observed in RNA viruses, the benchmark of variable populations (10). HCMV thus exists within hosts as a heterogeneous collection of diverse but related genomes, herein termed a population, rather than a single sequence.…”

“…HCMV thus exists within hosts as a heterogeneous collection of diverse but related genomes, herein termed a population, rather than a single sequence. The high levels of intrahost genetic diversity in HCMV infections are seen across a range of human hosts, from healthy adults (14) and children (18) to congenitally infected infants (19), transplant recipients (17), and AIDS patients (20). The clinical importance of HCMV genetic diversity is still debated (21), but increasing evidence suggests a correlation with HCMV pathogenesis.…”

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

“…A recent analysis of the genomic variability of CMVs isolated from congenitally infected neonates indicates that CMV exists within its host as a mixture of genetically diverse genomes (118)(119)(120). Recent efforts have employed high-throughput sequencing to study the extent of genome-wide variability in clinical isolates (121). Remarkably, intrahost CMV populations were found to be as variable as some RNA virus quasispecies, and that CMV diversity spanned the entire length of the genome, including the genes for the envelope proteins present on the CMV membrane (118).…”

Quantitative Characterization of Defective Virus Emergence by Deep Sequencing

“…High-throughput (deep) sequencing has been widely used to characterize the diversity of virus variants in natural infections as well as their sequence level adaptations to antiviral drugs (27)(28)(29)(30)(31)(32)(33)(34)(35).…”