Virome Variation during Sea Star Wasting Disease Progression in Pisaster ochraceus (Asteroidea, Echinodermata)

Abstract

Sea star wasting disease (SSWD) is a condition that has affected asteroids for over 120 years, yet mechanistic understanding of this wasting etiology remains elusive. We investigated temporal virome variation in two Pisaster ochraceus specimens that wasted in the absence of external stimuli and two specimens that did not experience SSWD for the duration of our study, and compared viromes of wasting lesion margin tissues to both artificial scar margins and grossly normal tissues over time. Global assembly of all SSWD-affected tissue libraries resulted in 24 viral genome fragments represented in >1 library. Genome fragments mostly matched densoviruses and picornaviruses with fewer matching nodaviruses, and a sobemovirus. Picornavirus-like and densovirus-like genome fragments were most similar to viral genomes recovered in metagenomic study of other marine invertebrates. Read recruitment revealed only two picornavirus-like genome fragments that recruited from only SSWD-affected specimens, but neither was unique to wasting lesions. Wasting lesion margin reads recruited to a greater number of viral genotypes (i.e., richness) than did either scar tissue and grossly normal tissue reads. Taken together, these data suggest that no single viral genome fragment was associated with SSWD. Rather, wasting lesion margins may generally support viral proliferation.

Keywords: asteroidea; densovirus; nodavirus; picornavirus; sea star wasting disease.

Figure 1

Gross examination of SSWD lesion on a Pisaster ochraceus specimen retrieved from Davenport, CA at the time of sampling for this survey. A = grossly normal tissue; B = lesion margin; C = lesion (underlying body wall tissues); D = papula and pedicellaria; E = paxilla (spine).

Figure 2

Gross changes in Pisaster ochraceus observed in specimens used in viral metagenome analyses over time, and detail (indicated by arrows) of wasting lesion and artificial scars sampled. The elapsed time is indicated on each panel.

Figure 3

Maps of contiguous sequences matching Picornavirales recovered in this survey of Pisaster ochraceus. Contigs were annotated based on BLASTx (e-value < 1 × 10⁻²⁰) against the non-redundant database at NCBI. The color of arrows (open reading frames) indicates the taxonomic identity of their best matches. Numbers above the ORFs indicate the e-value of BLAST results. The total contig lengths are indicated by solid lines running through and between ORFs.

Figure 4

Phylogenetic representations of Picornavirales-like genome fragments recovered from Pisaster ochraceus. The cladograms were constructed based on a 98 amino acid alignment of the RNA dependent RNA polymerase gene (A), a 187 amino acid alignment of the rhv-like capsid domain (B), a 160 amino acid alignment of the RNA helicase domain (C) and 193 amino acid of the RNA dependent RNA polymerase gene (D,E) and performed separately for overlapping regions including best matches at NCBI. Trees were constructed by Neighbor Joining and based on Jukes-Cantor distance in the CLC Genomics Workbench 4.0 (Qiagen). Bootstrap values >50% (based on 1000 iterations) are indicated above nodes. The host identity is indicated by symbols next to branch labels. An additional phylogenetic representation of each tree based on maximum likelihood is presented in Figure S3.

Figure 5

Phylogenetic representations of Nodamuvirales-like genome fragments recovered from Pisaster ochraceus. The trees were constructed based on a 101 amino acid (A) and 559 amino acid (B) alignments of the RNA dependent RNA polymerase gene of the nodavirus RNA1 genome fragment including best matches at NCBI. Trees were constructed by Neighbor Joining and based on Jukes-Cantor distance using CLC Genomics Workbench 4.0 (Qiagen). Bootstrap values >50% (based on 1000 iterations) are indicated above nodes. The host identity is indicated by symbols next to branch labels. An additional phylogenetic representation of each tree based on maximum likelihood is presented in Figure S5.

Figure 6

Map of densovirus-like genome fragments recovered from Pisaster ochraceus from Davenport, CA in July 2018. The length of contig is given by the solid black line running through open reading frames (ORFs; indicated by arrows). The color of arrow indicates the top BLASTx match to the non-redundant database at NCBI, and e-value of the match given above each ORF.

Figure 7

Phylogenetic representations of Densovirinae-like genome fragments recovered from Pisaster ochraceus. The cladorgrams were constructed based on: a 103 amino acid alignment of the structural (coat protein) gene; a 83 amino acid of the non-structural 1 (NS1) gene; a 112 amino acid of the NS2 gene; and a 111 amino acid of the NS3 gene. Phylogenetic representations include best matches by BLASTx against the non-redundant database at NCBI. Trees were constructed by Neighbor Joining and based on Jukes-Cantor distance using the CLC Genomics Workbench 4.0 (Qiagen). Bootstrap values >50% (based on 1000 iterations) are indicated above nodes. The host identity is indicated by symbols next to branch labels. An additional phylogenetic representation of each tree based on maximum likelihood is presented in Figure S6.

Figure 8

Heat map of viral contig read recruitment across all libraries in non-wasted and wasted asteroids. Dark cells = viral contig recruited reads from library, white cells = viral contig did not recruit reads from library. T0 = initial sample, Ti = time of lesion formation, Tf = experiment terminaton. C = grossly normal tissue, S = artificial scar tissue, L = wasting lesion margin. Phylogeny determined by family-level assignment based on nearest relative match (BLASTx) against non-redundant (nt) database at NCBI.

Figure 9

Richness of viral genome fragments recruiting reads from SSWD-affected and grossly normal tissues in viral metagenomes prepared from P. ochraceus during temporal study of wasting. Significance (a,b) determined by Student’s t-test (p < 0.008, df = 4 with Bonferroni correction for 6 tests). T0 = initial sample, Ti = time of first lesion appearance, Tf = experiment termination. Open squares represent mean (error bars = SE).