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

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 However, analysis of different samples withdrawn from the same patients at different time points revealed that the relative levels of replication of the individual HCMV genotypes changed within a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

Differences in Growth Properties among Two Human Cytomegalovirus Glycoprotein O Genotypes

Glycoprotein O (gO) of the human cytomegalovirus (HCMV) is the critical subunit of the envelope trimer gH/gL/gO as it interacts with platelet-derived growth factor alpha receptor upon fibroblast entry, and triggers gB-mediated fusion for fibroblast and epithelial cell infection. Eight genotypes (GT) of the highly polymorphic gO gene are described, yet it is unclear whether the distinct GTs differ in their function. Thus, we aimed to elucidate potential functional differences between two highly diverse gO GTs in an otherwise genomically identical HCMV strain. Therefore, resident gO GT1c sequence of strain TB40-BAC4-luc was entirely replaced by gO GT4 of strain Towne and both, GT1c and GT4 viruses, were investigated for their growth properties in fibroblasts and epithelial cells. In addition, two conserved gO cysteines involved in gH/gL/gO stabilization were mutated to serine either in GT1c (C218S and C343S) or GT4 (C216S and C336S) and their effects on cell-free infectivity were assessed. GT4 viruses displayed a significantly enhanced epithelial cell tropism and this resulted in higher virus release upon replication in epithelial cells when compared to GT1c viruses. Further, when the two cysteines were individually mutated in gO GT1c no impairment in cell-free infectivity was observed. This, however, was in sharp contrast to gO GT4, in which both of the corresponding cysteine mutations led to a substantial reduction in cell-free infectivity which was even more pronounced upon mutation of GT4-C336 than of GT4-C216. In conclusion, these findings provide evidence that the two highly diverse gO genotypes, GT1c and GT4, differ in their functional properties as revealed by their different infection capacities for epithelial cells and by their different responsiveness to mutation of strictly conserved cysteine residues. Thus, it is likely that the gO heterogeneity influences cell-free infectivity of HCMV also in vivo which may have important implications for virus host transmission

Pre-transplant plasma Torque Teno virus load and increase dynamics after lung transplantation.

The human Torque Teno virus (TTV) causes persistent viremia in most immunocompetent individuals. Elevated TTV levels are observed after solid organ transplantation and are related to the extent of immunosuppression especially during the phase of maintenance immunosuppression. However, the extent to which the TTV increase in the early phase post-transplantation is associated with the patient's immunosuppressive state is unclear.In this study, we assessed the TTV increase dynamics in detail during the first three months after lung transplantation under a defined immunosuppressive regimen and in relation to the pre-transplant TTV level.Forty-six lung transplant recipients (LTRs) were included in this prospective longitudinal study. All received alemtuzumab induction combined with tacrolimus and corticosteroids immunosuppressive therapy. Plasma TTV DNA was monitored before transplantation and regularly within the first three months post-transplantation (n = 320 samples; mean sampling interval: 12.2 days).In 43/46 LTRs (93%), TTV DNA was detectable before transplantation (median 4.4 log10 copies/mL; range: 2.0-6.4). All 46 LTRs showed a TTV increase post-transplantation, which followed a sigmoidal-shaped curve before the median peak level of 9.4 log10 copies/mL (range: 7.6-10.7) was reached at a median of day 67 (range: 41-92). The individual TTV DNA doubling times (range: 1.4-20.1 days) significantly correlated with the pre-transplant TTV levels calculated over 30 or 60 days post-transplantation (r = 0.61, 0.54, respectively; both P < 0.001), but did not correlate with the mean tacrolimus blood levels. Pre-transplant TTV levels were not associated with time and level of the patients' post-transplant TTV peak load.The TTV level may be used to mirror the state of immunosuppression only after the patients' initial peak TTV level is reached

Temporal dynamics of the lung and plasma viromes in lung transplant recipients

<div><p>The human virome plays an important role for the clinical outcome of lung transplant recipients (LTRs). While pathogenic viruses may cause severe infections, non-pathogenic viruses may serve as potential markers for the level of immunosuppression. However, neither the complexity of the virome in different compartments nor the dynamics of the virus populations posttransplantation are yet understood. Therefore, in this study the virome was analyzed by metagenomic sequencing in simultaneously withdrawn bronchoalveolar lavage (BAL) and plasma samples of 15 LTRs. In seven patients, also follow-up samples were investigated for abundance and dynamics of virus populations posttransplantation. Five eukaryotic and two prokaryotic virus families were identified in BAL, and nine eukaryotic and two prokaryotic families in plasma. Anelloviruses were the most abundant in both compartments, followed by Herpes- and Coronaviruses. Virus abundance was significantly higher in LTRs than in healthy controls (Kruskal-Wallis test, <i>p</i><0.001). Up to 48 different anellovirus strains were identified within a single LTR. Analyses in the follow-up patients revealed for the first time a highly complex and unique dynamics of individual anellovirus strains in the posttransplantation period. The abundance of anelloviruses in plasma was inversely correlated with that of other eukaryotic viruses (Pearson correlation coefficient <i>r</i> = −0.605; <i>p<</i>0.05). A broad spectrum of virus strains co-exists in BAL and plasma of LTRs. Especially for the anelloviruses, a high degree of co-infections and a highly individual and complex dynamics after transplantation was observed. The biological impact of these findings and their relation to clinical variables remain to be elucidated by future analyses.</p></div

General information of the 15 lung transplant recipients included in this study.

<p>General information of the 15 lung transplant recipients included in this study.</p

Relation between the abundance of anelloviruses and of other eukaryotic viruses.

<p>(A) and (B) Abundance of anelloviruses (Log10) versus the abundance of other eukaryotic viruses (non-anelloviruses) in BAL and plasma of 15 LTRs, respectively. Only one sample per patient was used for this analysis, for the time points closer to 87 days after transplantation.</p

Viral families identified in 15 lung transplant recipients.

<p>(A) Viral families identified in BAL samples, and (B) plasma samples obtained simultaneously at different time points after transplantation (F1-F7: LTRs with follow up samples; P1-P8: LTRs tested at only one time point). (C) Viral families identified in 7 healthy controls (C1-C7). Numbers after the patient identifier indicate days after transplantation for LTRs; and days between first and second sample for the healthy controls. Log10 abundance in reads per million.</p

Within patient anellovirus isolates present at each time point after transplantation in BALs and plasma samples of LTRs (F1-F3).

<p>C2 is a healthy control sample. *For C2, y-axis indicates day at which sample was taken. Blue lines: anellovirus strains detected simultaneously in BAL and plasma. Black lines: anellovirus strains present in only one body compartment.</p

Abundance and diversity of anellovirus strains in BAL and plasma.

<p>(A) and (B) Average abundance of anellovirus strains found over all time points in BAL and plasma samples of LTRs. The strains are shown according to phylogenetic similarity and grouped by genogroup. Bar height indicates Log10 abundance (reads per million). (C) Shannon diversity indexes of LTR BAL samples based on TTV strains. (D) Shannon diversity indexes of LTR plasma samples based on TTV strains.</p

Abundance of anelloviruses in different body compartments and number of strains by genogroup.

<p>(A) Abundance of anelloviruses (number of sequencing reads per million) in BAL and plasma of 15 LTRs and in plasma of healthy controls (***:<i>p</i><0.001; Kruskal-Wallis with post-hoc test). (B) Number of anellovirus strains in each genogroup normalized by the total number of reads sequenced for each sample and the total number of different strains identified per genogroup (*:<i>p</i><0.05; Wilcoxon rank sum test).</p