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 However, analysis of different samples withdrawn from the same patients at different time points revealed that the relative levels of replication of the individual HCMV genotypes changed within a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.

FIG. 1.

Relative abundances of gN, gO, and UL139 genotype populations determined by UDPS. (A) Schematic representation of the highly polymorphic regions analyzed. Nucleotide numbers are based on the open reading frames (ORFs) of the AD169 gene sequence (GenBank accession number X17403.1). (B) Illustration of the number and quantitative relationship of the individual gN, gO, and UL139 genotypes present in the 12 samples indicated on the x axis. Patient samples are numbered as described in Table 1, with “B” and “P” indicating BAL samples and plasma, respectively. The genotype frequencies are percentages of the total viral DNA load. *, number of genotypes detected; brackets, paired BAL and plasma samples.

FIG. 2.

Phylogenetic analysis of gN, gO, and UL139 genotypes. Shown are neighbor-joining trees constructed using all unique genotype sequences for each genomic region found in the 12 patient samples. The trees illustrate both the frequency of the genotypes as a percentage of the total viral DNA load and their clustering with known prototype genotype sequences, as shown by boldface and gray shading. Genotype designations are used as reported previously (3, 24, 30, 31, 35, 45). Patient samples are numbered as described in Table 1.

FIG. 3.

Quantitative distribution and nucleotide sequence stability of gO genotype populations at different times posttransplantation. (A to D) The relative frequency of each gO genotype as a percentage of the total viral DNA load for each of the four lung transplant patients at each time point tested is displayed. (E) Neighbor-joining tree inferred from an alignment of unique gO genotype sequences obtained for each patient at different time points. Patient samples, numbered as described in Table 1, are followed by the times of sampling in months posttransplantation (post-Tx).