The Perioperative Lung Transplant Virome: Torque Teno Viruses Are Elevated in Donor Lungs and Show Divergent Dynamics in Primary Graft Dysfunction

Abstract

Primary graft dysfunction (PGD) is a principal cause of early morbidity and mortality after lung transplantation, but its pathogenic mechanisms are not fully clarified. To date, studies using standard clinical assays have not linked microbial factors to PGD. We previously used comprehensive metagenomic methods to characterize viruses in lung allografts >1 mo after transplant and found that levels of Anellovirus, mainly torque teno viruses (TTVs), were significantly higher than in nontransplanted healthy controls. We used quantitative polymerase chain reaction to analyze TTV and shotgun metagenomics to characterize full viral communities in acellular bronchoalveolar lavage from donor organs and postreperfusion allografts in PGD and non-PGD lung transplant recipient pairs. Unexpectedly, TTV DNA levels were elevated 100-fold in donor lungs compared with healthy adults (p = 0.0026). Although absolute TTV levels did not differ by PGD status, PGD cases showed a smaller increase in TTV levels from before to after transplant than did control recipients (p = 0.041). Metagenomic sequencing revealed mainly TTV and bacteriophages of respiratory tract bacteria, but no viral taxa distinguished PGD cases from controls. These findings suggest that conditions associated with brain death promote TTV replication and that greater immune activation or tissue injury associated with PGD may restrict TTV abundance in the lung.

Keywords: immunosuppression/immune modulation; lung (allograft) function/dysfunction; lung failure/injury; lung transplantation/pulmonology; microbiomics; molecular biology; translational research/science.

Figure 1. Torque Teno Virus Levels in Lung Transplant Donors, Recipients and Healthy Adults

(A) Torque teno viruses quantified by qPCR in BAL. Boxes represent the middle two quartiles for each group, with the bold line representing the median value. Dots represent individual samples. Donor BAL was taken prior to organ procurement. Recipient BAL and Recipient serum were taken one hour after organ reperfusion. Quantities of TTV were higher in Donor and Recipient BAL compared to BAL of healthy adults as determined by Wilcoxon Rank Sum test (p=0.0026 and p<0.001, respectively). (B) Quantities of TTV in lung transplant recipient serum were lower compared to healthy adults as determined by Wilcoxon Rank Sum test (p<0.001). (C) Log10 TTV levels in paired Donor and Recipient BAL from individual samples were correlated (p<0.001, Spearman’s rho=0.548. A linear model was fitted to the data and is shown by the black line. The limit of quantification for the qPCR assay ranged from 11–65 copies/reaction. **p<0.01, ***p<0.001

Figure 2. Torque Teno Virus Dynamics in Perioperative Period and Association with Primary Graft Dysfunction

(A) TTV levels from organ pre-procurement (Donor) and post-reperfusion (Recipient) BAL samples are shown for each PGD case and control. Samples from the same organ are connected by a line. (B) The fold change in viral levels in the lung for PGD cases and controls is shown. Empty circles represent individual samples and the filled diamond represents the mean of the group. Both the average and median fold change was lower in PGD cases compared to paired controls (p=0.046 and p=0.041; Paired Student’s T-test and Wilcoxon Signed-Rank test, respectively). The number of paired samples analyzed was 15 due to missing samples for some patients.

Figure 3. The Perioperative Lung Viral Microbiome

Displayed is the number of shotgun metagenomic reads of DNA and cDNA libraries from each sample matching known viruses. Each column represents a different BAL sample. Each row represents a viral taxonomy at the family level, or at the species level for those hits that could not be classified into established families (species-level assignments for all viruses are shown in Fig. S2). Sequencing, processing of reads, alignments to viral genomes and removal of spurious hits was carried out as described in Methods. Columns are grouped by PGD-control pairs and labeled according to subject group, pair number and sample type as shown by the color coding across the top of the figure. Results of standard bacterial culture in each sample for the most commonly identified bacteria are also shown annotated on top. Further information on viral type and host is given by the column at the left. The intensity in each block represents the number of reads of each viral family in each sample on a log10 scale.

Figure 4. Diversity of Anellovirus Assignments in Lung and Serum During the Perioperative Period

(A) Displayed are filtered metagenomic reads from perioperative BAL samples aligning to annotated human Anelloviruses. Each column corresponds to an individual sample and each row corresponds to the top scoring reference Anellovirus genome in the NCBI Viral Database. The intensity of each block represents the number of reads from that sample aligning to that reference species. Columns are grouped by PGD-control pairs and labeled according to subject group, pair number and sample type. (B) Displayed are filtered metagenomic reads from post-transplant recipient serum that aligned to human Anellovirus entries in the NCBI Viral Database. In serum, all viral hits remaining after stringent filtering steps were to Anellovirus family members. Samples that had insufficient DNA after library preparation for Illumina sequencing are omitted.