Irene Görzer

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 However, analysis of different samples withdrawn from the same patients at different time points revealed that the relative levels of replication of the individual HCMV genotypes changed within a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

Emergence of Multiple Cytomegalovirus Strains in Blood and Lung of Lung Transplant Recipients

Background. Cytomegalovirus (CMV) is a major pathogen in lung transplant recipients (LTRs). The emergence of different CMV strains in lung and blood after transplantation has not yet been analyzed. Methods. In total, 75 serum and 91 broncheoalveolar lavage (BAL) samples obtained from 25 LTRs in the follow-up after transplantation were tested for the presence of different CMV strains. The gB, gN, and gO genes of the CMV isolates were analyzed by subtype-specific PCR, restriction fragment length polymorphism (RFLP), sequencing, and phylogenetic analysis. Results. Mixed CMV-strain populations were detected after cessation of antiviral prophylaxis in up to 80% and 90% of the patients’ BAL and serum, respectively, and this was independent of the CMV serostatus of donor and recipient. In five patients, the same single CMV strain was consistently detectable over at least 1 year in lung and blood, although in two of these cases donor and recipient had both been CMV-seropositive. Most CMV strains were distributed in the lung and blood compartment. Symptomatic CMV infection within the first year after transplantation was observed only in patients with mixed CMV-strain populations (P<0.05). Conclusion. Most LTRs harbor more than one CMV strain in their lung and blood compartment after cessation of prophylaxis, but the CMV strain distribution within and between the compartments varies between individuals and is not associated with the donor/recipient serostatus. The data further show that compartmentalization of CMV strains in lung versus blood seems to be a rare event and that the presence of mixed CMV-strain infections within the first year after transplantation may be disadvantageous for LTRs.

The impact of PCR-generated recombination on diversity estimation of mixed viral populations by deep sequencing

Ultra-deep pyrosequencing (UDPS) of targeted amplicons allows to determine a large number of individual sequence reads from a single PCR product, and this approach is thus extremely valuable for analysis of mixed viral populations. A mixture of genetically distinct DNA templates, however, may lead to the generation of recombinant sequences during the initial PCR amplification step. In the present study, the frequency at which these misleading PCR artefacts are formed has been estimated by using artificial mixtures of genetically distinct human cytomegalovirus (HCMV) DNA templates for a given cycling profile. The presence of similar copy numbers of non-identical HCMV target sequences, combined with high levels of input HCMV DNA, as is found in some clinical samples, favored the formation of recombinant PCR products. Thus, to estimate the full natural diversity within mixed viral populations using UDPS, artificial chimeras generated during the PCR step should be taken into account as a potential artefact.

The nuclear actin-related protein Act3p/Arp4p of Saccharomyces cerevisiae is involved in transcription regulation of stress genes

A mutational analysis of the essential nuclear actin‐related protein of Saccharomyces cerevisiae, Act3p/Arp4p, was performed. The five residues chosen for substitution were amino acids conserved between actin and Act3p/Arp4p, the tertiary structure of which most probably resembles that of actin. Two thermosensitive (ts) mutants, a single and a double point mutant, and one lethal double point mutant were obtained. Both ts mutants were formamide‐sensitive which supports a structural relatedness of Act3p/Arp4p to actin; they were also hypersensitive against hydroxyurea and ultraviolet irradiation pointing to a possible role of Act3p/Arp4p in DNA replication and repair. Their ‘suppressor of Ty’ (SPT) phenotype, ob‐served with another ts mutant of Act3p/Arp4p before, suggested involvement of Act3p/Arp4p in transcription regulation. Accordingly, genome‐wide expression profiling revealed misregulated transcription in a ts mutant of a number of genes, among which increased expression of various stress‐responsive genes (many of them requiring Msn2p/Msn4p for induction) was the most salient result. This provides an explanation for the mutants enhanced resistance to severe thermal and oxidative stress. Thus, Act3p/Arp4p takes an important part in the repression of stress‐induced genes under non‐stress conditions.

The nuclear actin-related protein Act3p/Arp4p is involved in the dynamics of chromatin-modulating complexes

Chromatin remodelling and histone‐modifying complexes govern the modulation of chromatin structure. While components of these complexes are diverse, nuclear actin‐related proteins (Arps) have been repeatedly found in these complexes from yeast to mammals. In most cases, Arps are required for functioning of the complexes, but the molecular mechanisms of nuclear Arps have as yet been largely unknown. The Arps and actin, sharing a common ancestor, are supposed to be highly similar in the three‐dimensional structure of their core regions, including the ATP‐binding pocket. The Arp Act3p/Arp4p of Saccharomyces cerevisiae exists within the nucleus, partly as a component of several high molecular mass complexes, including the NuA4 histone acetyltransferase (HAT) complex, and partly as uncomplexed molecules. We observed that mutations in the putative ATP‐binding pocket of Act3p/Arp4p increased its concentration in the high molecular mass complexes and, conversely, that an excess of ATP or ATPγS led to the release of wild‐type Act3p/Arp4p from the complexes. These results suggest a requirement of ATP binding by Act3p/Arp4p for its dissociation from the complexes. In accordance, a mutation in the putative ATP binding site of Act3p/Arp4p inhibited the conversion of the NuA4 complex into the smaller piccoloNuA4, which does not contain Act3p/Arp4p and exhibits HAT activity distinct from that of NuA4. Although the in vitro binding activity of ATP by recombinant Act3p/Arp4p was found to be rather weak, our observations, taken together, suggest that the ATP‐binding pocket of Act3p/Arp4p is involved in the function of chromatin modulating complexes by regulating their dynamics. Copyright

Plasma DNA levels of Torque teno virus and immunosuppression after lung transplantation

to nadir 3.39 1.1 liters/min (p o 0.001) and PI by mean 30.5% (range 12.1% to 62.5%) to 3.41 0.9 (p o 0.001) (Figure 1). The 2 patients with failed first shock therapies had the longest times in VF, respectively, at 11.3 and 12.0 seconds, associated with 34.1% and 64.4% decreases in flow output and PI at 34.1% and 62.5%, respectively. There was a correlation between time in VF and reduction in flow output (R 1⁄4 0.607, p 1⁄4 0.004) and PI (R 1⁄4 0.725, p o 0.001), respectively. All patients recovered baseline flow output after successful defibrillation. The mean shock energy was 25 8.6 J (range 10 to 40 J). The mean time to return to baseline output after successful defibrillation was 18 4 seconds (range 13 to 29 seconds). Regression analysis revealed no relationships between the number of testing events, time in VF and recovery times. The principal finding of this study is that ventricular fibrillation in the LVAD population is associated with a 32% mean decrease in flow output that was promptly restored to baseline by ICD therapy within 30 seconds in all cases. In the context of prior data, these findings further dispel the notion that patients on LVAD support are unaffected by ventricular tachyarrhythmias as once believed. This is highly relevant in an era in which patients on LVAD support are increasingly out of hospital awaiting cardiac transplantation or outpatient destination therapy. The availability of effective treatments, including a growing role for catheter ablation, further increases the importance of the present findings as it represents a treatment opportunity. The nadir flow outputs in our analysis typically dropped into the range of 3 liters/min, but fell as low as 1.6 liters/min. Caution is advised in interpretation of absolute values given the imperfect correlation with hemodynamic data. However, each patient in the study served as their own control and a significant drop occurred in every study patient on each VF event with time in VF correlating with the nadir in flow output. Such increasingly lower perfusion could result in end-organ compromise or thrombus formation at the apical inflow cannulae, as reported previously. Ventricular fibrillation in the LVAD population is associated with a 32% decrease in flow output, which can be promptly restored by ICD therapy. This may relate to the apparent ICD survival benefit in this population.

Virus Load Dynamics of Individual CMV-Genotypes in Lung Transplant Recipients With Mixed-Genotype Infections

Human cytomegalovirus (CMV) is a major cause of disease and transplant dysfunction in lung transplant recipients. Simultaneous emergence of more than one CMV‐genotype can occur, and appears to be disadvantageous for the patient. In this study, the dynamics of individual CMV‐genotypes in blood and lung was assessed within mixed CMV‐genotype populations emerging after lung transplantation. In 69 plasma and 76 bronchoalveolar lavage samples of 16 lung transplant recipients with mixed CMV‐genotype infections within the first year posttransplantation each of the major glycoprotein B (gB) and glycoprotein H (gH) genotypes was selectively quantified by genotype‐specific quantitative TaqMan assays. The data obtained revealed that individually different genotype dynamics occurred for the individual patients and that the relative levels of the genotypes to each other may change over time. The quantitative development was independent of the specific gB–gH‐genotype. In 10 of the 16 lung recipients the patients individual genotype composition was the same in blood and lung. Genotype development during the follow‐up was influenced by antiviral treatment. These data show for the first time that the CMV load used as diagnostic tool after transplantation is not always a constant entity but reflects the sum of the individual CMV‐genotype dynamics developing over time. J. Med. Virol. 80:1405–1414, 2008.

Human cytomegalovirus (HCMV) genotype populations in immunocompetent individuals during primary HCMV infection.

BACKGROUND Immunocompetent individuals can harbor multiple human cytomegalovirus (HCMV) genotypes. However, little is known about the genotype populations acquired during primary HCMV infection. OBJECTIVE The aim of this study was to assess the HCMV genotype populations present in the blood of non-immunocompromised patients experiencing primary HCMV infection. STUDY DESIGN HCMV glycoprotein B (gB), glycoprotein H (gH), and UL10 genotyping was performed on HCMV-positive serum samples of 36 immunocompetent patients during primary infection by sensitive gB- and gH-genotype-specific real-time-PCR assays and by UL10 sequencing. RESULTS In all cases only one gB-gH-UL10 genotype was detected. In contrast, mixed-genotype infections were found in 4 of 14 immunocompetent patients experiencing HCMV reactivation/reinfection (P=0.004). CONCLUSION Thus, the data support the presumption that multiple HCMV genotypes in immunocompetent individuals are often a result of serial reinfection rather than primary coinfection with different strains.

Pre-Transplant Plasma Torque Teno Virus Load and Increase Dynamics after Lung Transplantation

Background 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. Objectives 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. Study Design 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). Results 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. Conclusion The TTV level may be used to mirror the state of immunosuppression only after the patients’ initial peak TTV level is reached.

Analysis of the variability of CMV strains in the RL11D domain of the RL11 multigene family

Human cytomegalovirus (CMV) contains one of the largest genomes within the herpesvirus family and includes 12 multigene families. One of these is the RL11 family, whose members encode a characteristic domain, called RL11D. In the present study, the sequence variability of RL11D within the UL1, UL4, UL6, UL7, and UL10 genes of the RL11 family was investigated. For this purpose, these genes were analyzed in 70 clinical isolates obtained from urine, bronchoalveolar lavage, and blood of different patients. Substantial genetic variability among the clinical isolates was observed in all five genes analyzed. Based on phylogenetic analysis of variations in RL11D, the clinical isolates could be classified into three genotypes for UL1, 7, and 10 and, four genotypes for UL4 and 6. Further analysis showed statistically significant linkages between the following pairs of genes: UL6/UL7, UL4/UL7, UL1/UL4, and UL4/UL6. The data show that CMV strains exhibit a high interstrain variability in the RL11D domain of various RL11 family genes. Sequence variations, however, can be clearly grouped into a limited number of distinct genotypes. The genetic linkages found probably reflect a low frequency of recombination between genes that are arranged in close proximity on the viral genome.