First Report on Detection and Complete Genomic Analysis of a Novel CRESS DNA Virus from Sea Turtles

Abstract

To date, only a handful of viruses have been identified in sea turtles. Although eukaryotic circular Rep (replication initiation protein)-encoding single-stranded DNA (CRESS DNA) viruses have been reported from a wide variety of terrestrial species, and some of these viruses have been associated with clinical conditions in certain animals, limited information is available on CRESS DNA viruses from marine life. The present study aimed to investigate the presence of CRESS DNA viruses in sea turtles. In the present study, two (samples T3 and T33) of the 34 cloacal samples from 31 sea turtles (found in ocean waters around the Caribbean Islands of St. Kitts and Nevis) tested positive for CRESS DNA viruses by a pan-rep nested PCR assay. The partial Rep sequence of T3 shared 75.78% of a deduced amino acid (aa) identity with that of a CRESS DNA virus (classified under family Circoviridae) from a mollusk. On the other hand, the complete genome (2428 bp) of T33 was determined by an inverse nested PCR assay. The genomic organization of T33 mirrored those of type II CRESS DNA viral genomes of cycloviruses, characterized by the putative "origin of replication" in the 5'-intergenic region, and the putative Capsid (Cap)- and Rep-encoding open reading frame on the virion-sense- and antisense-strand, respectively. The putative Rep (322 aa) of T33 retained the conserved "HUH endonuclease" and the "super 3 family helicase" domains and shared pairwise aa identities of ~57% with unclassified CRESS DNA viruses from benthic sediment and mollusks. Phylogenetically, the T33 Rep formed a distinct branch within an isolated cluster of unclassified CRESS DNA viruses. The putative Cap (370 aa) of T33 shared maximum pairwise aa identity of 30.51% with an unclassified CRESS DNA virus from a capybara. Except for a blood sample from T33 that tested negative for CRESS DNA viruses, other tissue samples were not available from the sea turtles. Therefore, we could not establish whether the T3 and T33 viral strains infected the sea turtles or were of dietary origin. To our knowledge, this is the first report on the detection of CRESS DNA viruses from sea turtles, adding yet another animal species to the rapidly expanding host range of these viruses. Complete genome analysis of T33 identified a novel, unclassified CRESS DNA virus, providing insights into the high genetic diversity between viruses within the phylum Cressdnaviricota. Considering that sea turtles are an at-risk species, extensive studies on virus discovery, surveillance, and pathogenesis in these marine animals are of the utmost importance.

Keywords: Cressdnaviricota; complete genome analysis; novel CRESS DNA virus; sea turtle.

Figure 1

Geographical position of the Islands of St. Kitts and Nevis in the Caribbean region (Right). The map was adapted from https://www.cia.gov/library/publications/the-world-factbook (accessed on 1 April 2021). Map of St. Kitts and Nevis showing the locations (shown with colored stars) where the sea turtles were sampled or rescued after being stranded (Left). Black star: Shitten Bay; Blue star, Whitehouse Bay; Pink star, Timothy beach; Purple star, New River; Red star, South Friars beach. The map was adapted from https://www.google.com/maps (accessed on 2 February 2023).

Figure 2

Sea turtle T33, also known as Kayden. The sea turtle-associated CRESS DNA virus T33 was detected in a cloacal sample from Kayden.

Figure 3

The genomic organization of sea turtle-associated CRESS DNA virus T33. The open reading frames encoding the putative Capsid (Cap) and Replication initiation protein (Rep) are shown with red and blue arrows, respectively. The 5’- intergenic region of T33 contained the putative origin of replication on the Cap-encoding strand, which was characterized by the presence of a nonanucleotide motif (TAGTATTAC) at the apex of a well-defined stem-loop structure. The initiation codon for the putative Cap of T33 is shown with red font. The sizes of the Rep and Cap are shown in parentheses. nt: nucleotide; aa: amino acid.

Figure 4

The rolling circle replication (motifs I–III) and superfamily 3 helicase (Walker-A and -B, motif C, and arginine finger motif) motifs in the putative replication initiation proteins (Rep) of sea turtle-associated CRESS DNA virus T33 and other representative CRESS DNA viruses. Acidic, basic, hydrophobic, neutral, and polar amino acid residues are shown with red, blue, black, purple, and green color font, respectively. The numbers below the motif sequences of T33 correspond to the positions of the amino acid residue in the T33 Rep protein. The boxed section in Figure 4 was adapted from Figure 2 of Kazlauskas et al. [19] (licensed for free sharing and adaptation under the Creative Commons Attribution 4.0 International license, CC BY 4.0).

Figure 5

Phylogenetic analysis of the full-length deduced amino acid (aa) sequence of the putative replication initiation protein (Rep) of sea turtle-associated CRESS DNA virus T33 (highlighted with a red circle and indicated with a red arrow) with those of viruses representing the seven established viral families, six well-defined groups of unclassified CRESS DNA viruses (CRESSV1-6), two conserved groups of chimeric Reps (CRESS-Rec1 and -Rec2), and other unclassified CRESS DNA viruses within phylum Cressdnaviricota [18,19]. The virus name/source (detected in animal species)/country/year of sampling is shown for CRESS DNA virus T33, whilst the GenBank accession number/source (detected in animal species or environment)/classification status within phylum Cressdnaviricota have been mentioned for the other CRESS DNA viruses. Scale bar, 0.1 substitutions per aa residue. Bootstrap values of <60 are not shown. An enlarged image of the phylogenetic tree is shown in Supplementary Figure S4.