Redondoviridae, a Family of Small, Circular DNA Viruses of the Human Oro-Respiratory Tract Associated with Periodontitis and Critical Illness

Abstract

The global virome is largely uncharacterized but is now being unveiled by metagenomic DNA sequencing. Exploring the human respiratory virome, in particular, can provide insights into oro-respiratory diseases. Here, we use metagenomics to identify a family of small circular DNA viruses-named Redondoviridae-associated with human diseases. We first identified two redondovirus genomes from bronchoalveolar lavage samples from human lung donors. We then queried thousands of metagenomic samples and recovered 17 additional complete redondovirus genomes. Detections were exclusively in human samples and mostly from respiratory tract and oro-pharyngeal sites, where Redondoviridae was the second most prevalent eukaryotic DNA virus family. Redondovirus sequences were associated with periodontal disease, and abundances decreased with treatment. Some critically ill patients in a medical intensive care unit were found to harbor high levels of redondoviruses in respiratory samples. These results suggest that redondoviruses colonize human oro-respiratory sites and can bloom in several human disorders.

Keywords: CRESS DNA; brisavirus; critical illness; oro-respiratory virus; periodontitis; redondoviridae; redondovirus; vientovirus; viral metagenomics; virome.

Figure 1:. Discovery of Redondovirus Genomes in Metagenomic Samples

A-Several hundred shotgun metagenomic reads from two organ donor BAL virome samples were identified as having limited homology to Porcine stool-associated circular virus 5 (PoSCV-5). Reads from these samples were assembled into two contigs, which were then cloned from multiple displacement amplified sample DNA using target specific primers and Sanger sequenced. The complete circular genomes were used to query additional internal and public microbial metagenomic datasets. Target-specific amplification, sequencing and genome assembly was repeated for additional samples with sequences homologous to these novel genomes if the original DNA was available. In cases where original samples were not available, metagenomic contigs were checked for circularity and completeness. A total of 19 complete genomes were recovered from 67 human samples (bottom). See also Figure S1. B-The genomic architecture of redondoviruses shows ambisense open-reading frames (ORFs) encoding a conserved capsid, Replication associated protein (Rep) and unknown protein (ORF3). The average nucleotide identity of 20 Redondoviridae members (19 genomes discovered here and one genome previously reported (Cui et al., 2017)) is shown on the inside of the genome map as a heatmap. A putative origin of replication stem-loop structure with a conserved nonanucleotide motif is predicted to form in the 5’ end of the Rep coding region. The height of the letter in the motif represents its frequency.

Figure 2:. Redondoviridae is a Distinct Virus Family Based on Capsid and Rep Identities

Phylogenetic trees of redondovirus Rep (A) and capsid (B) proteins from CRESS DNA viruses. Collapsed viral genera or families are indicated by grey triangles. Branch likelihood, determined by approximate likelihood ratio test, is shown by colored circles at each node and the scale shows amino acid substitutions per site. The sample type of origin for each redondovirus is shown as colored boxes next to each virus’ name, which is colored to reflect genus designation. See also Table S2.

Figure 3:. Redondovirus Genomes Contain Conserved Motifs Implicated in Rolling-circle Replication

A-The sequence and predicted structure of the putative replication origin of Human lung-associated brisavirus AA is shown in the top left. The inverted repeat forming the stem is shown in orange, the nonanucleotide motif within the loop is shown in green, and an imperfect 6 bp direct repeat sequence is shown in purple. Individual predicted stem loop sequences (threshold for stability: ΔG°<−5 kcal/mol) are shown to the right of the folded sequence. The calculated ΔG° of melting for the predicted stem-loops ranges from −5.0 to −9.45 kcal/mol. B-Conserved rolling circle replication and superfamily 3 (SF3) helicase motifs were found in redondoviruses. The positions for the motifs are given using the Human lung-associated brisavirus AA genome sequence (Accession MK059754). The height of each letter represents its frequency. Amino acid positions identified as possibly under positive selection pressure are marked by a red star. C-The putative redondovirus capsid protein contains a basic amino-terminus and a predicted virus coat protein-like fold. The positions for the motifs correspond to Human lung-associated brisavirus AA, as above. Amino acid positions identified as possibly under positive selection pressure are marked by a red star.

Figure 4:. Frequency of Redondovirus Detection and Co-occurrence with Human DNA Viruses

A-Reads from 173 metagenomic datasets encompassing different human and non-human sample types were aligned to redondovirus genomes. A positive hit was determined based on 25% coverage of any redondovirus genome by short-read alignment. The percentage of samples that were positive is plotted on the y-axis and human body sites and other sample types are shown on the x-axis. The total number of samples analyzed in each category is annotated above and the total number of positive samples is indicated within the bar. See also Table S3. B-The clinical status breakdown, if available, of redondovirus-positive samples is shown. C-Reads from a subset of 20 datasets across 9 body sites (n = 2,675) were analyzed for homology to 20 redondovirus genomes and 133 animal-cell DNA viruses from six viral families. The height of each column represents the total number of samples that had detections of multiple viral families (rows). The viral families included in the co-detections are depicted as filled dots connected with lines below. The length of the bars on the left represents the total number of samples in which that viral family was detected. Cases where redondoviruses were detected are indicated in blue. See also Figure S2 and S3. IBD, inflammatory bowel disease

Figure 5:. Redondoviruses in the Oro-Respiratory Tract in Humans with Critical Illness and Periodontitis

A. Quantification of redondovirus genome sequences in post-SWGA DNA from oropharyngeal swabs (oropharynx) from 60 healthy volunteers, and oropharyngeal swabs and endotracheal aspirates (lung secretions) from 69 critically ill subjects. The average cycle of threshold (Ct) value of technical replicates is plotted on the y-axis. Samples with undetermined (i.e: no amplification) value in all 3 replicates are assigned an arbitrary value above the Ct value of the limit of resolution of the assay (37) which corresponds to 11 target copies per reaction. Samples below this value are counted as authentic detections. Negative controls included extraction blanks, reagent blanks and no template controls. Positive controls represent replicates of 10⁴ copies of Human lung-associated brisavirus RC spiked into DNA extracted from a redondovirus-negative lung sample, subjected to SWGA, and assayed by qPCR. B qPCR was used for redondovirus detection in respiratory and/or stool samples from 69 subjects in the medical intensive care unit (ICU). Six total subjects, 3 males and 3 females, were positive for redondoviruses. The time point and type of sample surveyed for these six subjects is shown on the x- and y-axis, respectively. Positive samples are indicated by a filled-circle and negative samples by an open circle. C-Number of reads mapping to a redondovirus in periodontitis samples from (Shi et al., 2015). Each point represents the average of all samples from a particular individual either before treatment (red) or after disease resolution (blue). Points from the same subject are connected by grey lines. The horizontal black line indicates the median. The Wilcoxon signed-rank test was used to test for paired differences between groups. D-Each point represents the number of reads mapping to a redondovirus in samples from (Califf et al., 2017) from subjects with periodontitis whose disease either did (blue) or did not (red) improve during the study. The horizontal black line indicates the median. The Wilcoxon rank-sum test was used to test for differences between groups. See also Table S4.