An Unconventional Flavivirus and Other RNA Viruses in the Sea Cucumber (Holothuroidea; Echinodermata) Virome

Abstract

Sea cucumbers (Holothuroidea; Echinodermata) are ecologically significant constituents of benthic marine habitats. We surveilled RNA viruses inhabiting eight species (representing four families) of holothurian collected from four geographically distinct locations by viral metagenomics, including a single specimen of Apostichopus californicus affected by a hitherto undocumented wasting disease. The RNA virome comprised genome fragments of both single-stranded positive sense and double stranded RNA viruses, including those assigned to the Picornavirales, Ghabrivirales, and Amarillovirales. We discovered an unconventional flavivirus genome fragment which was most similar to a shark virus. Ghabivirales-like genome fragments were most similar to fungal totiviruses in both genome architecture and homology and had likely infected mycobiome constituents. Picornavirales, which are commonly retrieved in host-associated viral metagenomes, were similar to invertebrate transcriptome-derived picorna-like viruses. The greatest number of viral genome fragments was recovered from the wasting A. californicus library compared to the asymptomatic A. californicus library. However, reads from the asymptomatic library recruited to nearly all recovered wasting genome fragments, suggesting that they were present but not well represented in the grossly normal specimen. These results expand the known host range of flaviviruses and suggest that fungi and their viruses may play a role in holothurian ecology.

Keywords: Apostichopus; flavivirus; holothurian; totivirus; virus; wasting.

Figure 1

Contig map of Apostichopus californicus flavivirus-like contig 91. The open reading frame matched a flavivirus polyprotein by BLASTx [37]. Methyltransferase, NS5 and helicase domains were identified by comparison against the conserved domain database (CDD) at NCBI [41]. The location of the envelope region was determined by protein folding comparison in Phyre [39]. The hairpin like structure preceding the Envelope region was determined by folding all sites between start (AUG) codons by mFold [40].

Figure 2

Phylogenetic representation of Apostichopus californicus flavivirus-like contig 91. The tree was constructed by performing an alignment of overlapping regions with best BLASTx matches at NCBI using the CLC Sequence Viewer 8.0 native alignment algorithm. The tree is based on a ~420 amino acid alignment by neighbor joining and based on Jukes-Cantor distance. Values above nodes indicate bootstrap statistics (>50%) based on 1000 iterations. The green branches indicate the emerging aquatic and invertebrate-associated flavivirus clade [52]. An additional phylogenetic representation based on maximum likelihood is provided in the Supplementary Materials.

Figure 3

Contig maps for Picornavirales-like genome fragments recovered from holothurians by viral metagenomics. The expect values of best matches by BLASTx against the non-redundant database at NCBI are indicated to the right of each contig. Solid lines indicate the total contig length, and arrows indicate open reading frames. Colored bars indicate shared homology between contigs based on reciprocal tBLASTx.

Figure 4

Phylogenetic representation of holothurian-associated Picornavirales-like genome fragments. The tree was constructed by performing an alignment of an overlapping region (~100 amino acid) of the rhv domain with best BLASTx matches in the non-redundant database at NCBI. The tree was constructed by neighbor joining and based on Jukes-Cantor distance. Values above nodes indicate bootstrap statistics (>50%) based on 1000 iterations. An additional phylogenetic representation based on maximum likelihood is provided in the Supplementary Materials.

Figure 5

Contig maps for totivirus-like genome fragments recovered from holothurians. The expected values of best matches by BLASTx [37] to the non-redundant database at NCBI are indicated to the right of the contigs. Solid lines indicate the total contig length, and arrows indicate open reading frames. RdRp = RNA-dependent RNA polymerase, Cp = capsid protein.

Figure 6

Phylogenetic representations of holothurian-associated totivirus-like genome fragments. The trees were constructed by performing an alignment of an overlapping region of the RdRp (top) and Cp (bottom) domains with best BLASTx matches at NCBI. The trees are based on ~156 amino acid (for RdRp) and 87 amino acid (for Cp) alignments by neighbor joining and based on Jukes-Cantor distance. Values above nodes indicate bootstrap statistics (>50%) based on 1000 iterations. An additional phylogenetic representation based on maximum likelihood is provided in the Supplementary Materials.