Viral metagenomics reveal blooms of anelloviruses in the respiratory tract of lung transplant recipients

Abstract

Few studies have examined the lung virome in health and disease. Outcomes of lung transplantation are known to be influenced by several recognized respiratory viruses, but global understanding of the virome of the transplanted lung is incomplete. To define the DNA virome within the respiratory tract following lung transplantation we carried out metagenomic analysis of allograft bronchoalveolar lavage (BAL), and compared with healthy and HIV+ subjects. Viral concentrates were purified from BAL and analyzed by shotgun DNA sequencing. All of the BAL samples contained reads mapping to anelloviruses, with high proportions in lung transplant samples. Anellovirus populations in transplant recipients were complex, with multiple concurrent variants. Quantitative polymerase chain reaction quantification revealed that anellovirus sequences were 56-fold more abundant in BAL from lung transplant recipients compared with healthy controls or HIV+ subjects (p < 0.0001). Anellovirus sequences were also more abundant in upper respiratory tract specimens from lung transplant recipients than controls (p = 0.006). Comparison to metagenomic data on bacterial populations showed that high anellovirus loads correlated with dysbiotic bacterial communities in allograft BAL (p = 0.008). Thus the respiratory tracts of lung transplant recipients contain high levels and complex populations of anelloviruses, warranting studies of anellovirus lung infection and transplant outcome.

Keywords: immunosuppression/immune modulation; lung transplantation/pulmonology; lung transplantation: living donor; microbiomics; molecular biology; translational research/science.

Figure 1. Overview of experimental design

BAL and OW samples (1–5 ml) were filtered through a 0.22 µM filter, concentrated on an Amicon 10 kDa MWCO filter, washed, and treated with DNase and RNase to yield purified virus-like particles. Select samples were checked for viral purity by quantifying the bacterial 16S rRNA and human β-tubulin genes using Q-PCR. Anelloviruses were quantified in all samples by Q-PCR. Select lung transplant and HIV+ samples were whole-genome amplified by multiple displacement amplification (MDA) and shotgun sequenced using the Illumina MiSeq platform. Bioinformatic analysis of the virome reads consisted of removing human reads (BMTagger) and aligning to the NCBI viral database. Reads were assembled into contigs, aligned to the NCBI viral database and analyzed.

Figure 2. Anelloviruses in BAL of HIV+ individuals and lung transplant recipients

A) Distribution of reads aligning to anelloviruses. Metagenomic sequencing reads were searched against the NCBI viral database and the number of combined reads aligning to each anellovirus calculated for lung transplant recipients (n=6) and HIV+ subjects (n=3). B) Presence of multiple anelloviruses present within the lungs of lung transplant recipient Tx-24 verified by Sanger sequencing. For each of the three analyzed by Sanger sequencing (x-axis), similarity was scored versus their closest matching NCBI reference sequence (y-axis). Open reading frames were assigned using MacVector and are illustrated on the genetic maps under the dot plots.

Figure 3. Diversity of anellovirus ORF1 sequences in samples from each individual studied

ORF1 amino acid sequences from anellovirus contigs and Genbank reference sequences were aligned and trimmed to generate the phylogenetic tree shown. The names of the reference sequences include the TTV strain name followed by the Genbank identification (gi) number. Clades are designated as groups, in the outer ring, as described by Okamoto et al (12, 22). The BAL sample of origin of each ORF1 sequence is designated by the color code (key at right). The Shimodaira-Hasegawa (SH) score was calculated using FastTree to estimate the reliability of each split compared to alternate topologies (25). Local SH support values over 0.9 are labeled as circles on the nodes of the tree. The scale bar represents the number of amino acid substitutions per position.

Figure 4. Abundance of anelloviruses in BAL samples

Anelloviruses were quantified by Q-PCR in BAL from lung transplant recipients, healthy individuals, and HIV+ individuals. Boxes represent the middle two quartiles for each group and whiskers are placed at the minimum and maximum values. Quantities were higher in lung transplant recipients compared with healthy and HIV+ individuals as determined by the Mann-Whitney test with Bonferroni correction: p<0.0002. All samples quantified were above the Q-PCR detection limit (1.4 copies per reaction).

Figure 5. Abundance of anelloviruses in OW samples

Anelloviruses were quantified by Q-PCR in oropharyngeal wash from healthy individuals and lung transplant recipients. Anellovirus quantities were higher in the upper respiratory tract of lung transplant recipients compared with healthy individuals. Mann-Whitney test: p=0.0055. All samples quantified were above the Q-PCR detection limit.

Figure 6. Comparison of anellovirus quantities in lung and upper respiratory tract within individuals

Paired Wilcoxon signed rank tests were performed on anellovirus copies from BAL and OW in A) healthy control subjects and B) lung transplant recipients. Anellovirus DNA copies were lower in the lung compared with the upper respiratory tract of healthy controls but not in lung transplant recipients (p-values: 0.0068 and 0.23, respectively).

Figure 7. Ordination based on composition of bacterial 16S sequence analysis, showing the relationship to anellovirus DNA copy numbers

Each circle on the plot shows data for a single transplant recipient BAL sample. For each sample, bacterial 16S rRNA gene tags were subject to deep sequencing (median 7,925 reads per sample; data published in (20)) and analyzed using Weighted UniFrac, which generated a set of pairwise distances among samples. The distances were then analyzed using Principal Coordinate Analysis (PCoA), and the samples plotted along the first two principal coordinates. For each sample, the anellovirus titer is summarized by the size of the open circle. Among these transplant recipient BALs there is a significant relationship between anellovirus DNA level and 16S bacterial community composition, which is reflected in the observation that samples with low anellovirus titers tend to appear on the lower left side of the PCoA plot (p=0.032; ADONIS test).