Genomic and morphological characterization of a novel iridovirus, bivalve iridovirus 1 (BiIV1), infecting the common cockle (Cerastoderma edule)

Abstract

High rates of mortality of the common cockle, Cerastoderma edule, have occurred in the Wash Estuary, UK, since 2008. A previous study linked the mortalities to a novel genotype of Marteilia cocosarum, with a strong correlation between cockle moribundity and the presence of M. cocosarum. Here, we characterize a novel iridovirus, identified by chance during metagenomic sequencing of a gradient purification of Marteilia cells, with the presence also correlated to cockle moribundity. The novel 179,695 bp iridovirus, bivalve iridovirus 1 (BiIV1), encodes 193 predicted ORFs and has a G+C content of 41 mol%. BiIV1 clusters together with other aquatic invertebrate iridoviruses in phylogenetic analyses and has a similar genome size to other invertebrate iridoviruses. Comparative analysis revealed that BiIV1 has lost three genes that were previously thought to be common amongst all iridoviruses but has also gained genes, potentially from horizontal transfer from its bivalve mollusc host(s). Electron microscopy showed 158 nm icosahedral virions present in the haemocytes of cockles, typical of those observed in host tissues infected with viruses of the family Iridoviridae. Prevalence of BiIV1 in moribund cockles was higher than that in apparently healthy cockles at most sites in the Wash Estuary, with up to 100% PCR prevalence in moribund cockles. Our findings provide the first genome for a bivalve-infecting iridovirus and identify a second bivalve-associated iridovirus in publicly available genomic datasets, adding to the knowledge of invertebrate iridovirus genomics and diversity.

Keywords: aquatic animal health; bivalve; cockle; emerging disease; iridovirus.

Fig. 1.. Circular map of the 179,695 bp BiIV1 genome. The outer scale is numbered clockwise in kilobases. The outer graph in black and grey depicts G+C content (mol%) across the genome. The second innermost track depicts predicted ORFs on the positive strand, and the innermost track depicts the predicted ORFs on the negative strand. Dark green blocks represent ORFs with homology to the six core genes identified by Toenshoff et al. [16], mid-green blocks represent ORFs with similarity to other iridoviruses, light blue blocks represent ORFs with similarity to other viruses and yellow blocks represent ORFs with predicted protein sequence similar to bivalve proteins. Purple blocks represent ORFs with similarity to bacterial-derived proteins, pink blocks depict ORFs that contained sequences consistent with known protein motifs and ORFs represented by grey blocks had no similarity to known proteins or protein motifs.

Fig. 2.. Genome size (kbp) plotted against G+C content (mol%) for a representative set of Iridoviridae genomes. Alphairidovirinae are represented by squares, with genera within this subfamily represented by different colours. Betairidovirinae are represented by circles, with the four defined genera within this subfamily, and the three species within no assigned genus (including BiIV1), represented by different colours. Within the Betairidovirinae subfamily, Anopheles minimus iridovirus (accession number KF938901) has the smallest genome size at 163,023 kbp, and Daphnia iridovirus 1 (accession number LS484712) has the largest at 288,858 kbp. G+C content within Betairidovirinae ranges between 27.75 (cricket iridovirus – accession number OK181107) and 48.75 mol% (carnivorous sponge-associated iridovirus – accession number ON887238).

Fig. 3.. Bayesian consensus tree constructed from a concatenated multiple amino acid alignment of major capsid protein, DNA-directed RNA polymerase II subunit Rpb2, putative A32-like packaging ATPase, putative CTD phosphatase-like protein, putative helicase protein, putative transcription elongation factor S-II-like protein from BiIV1, 113 other Iridoviridae, 5 Ascoviridae and 2 Marseilleviridae. The tree is rooted to the Marseilleviridae clade. Branch labels denote posterior probabilities, with black circles used when posterior probability=1.

Fig. 4.. Electron micrographs of a common cockle, C. edule, infected with BiIV1. (a) Two host cells infected with BiIV1, with the nuclei, N, showing condensed chromatin (CC), and mature and immature BiIV1 virions in the cytoplasm (Cyt). The left cell has a large electron-lucent assembly site (*) containing a few mature virions and many immature, developing virions. The right cell contains a paracrystalline array of virus particles. Scale bar=2 µm. (b) Higher magnification of the right cell, with numerous mature virions (black arrowheads), a few immature, developing virions (white arrowheads) and putative viral DNA prior to packing (white arrow). Scale bar=500 nm. (c) Higher magnification of the left cell, with mature virions (black arrowheads) and immature, empty and developing capsids (white arrowheads). Scale bar=500 nm. (d) High magnification of mature virions in a paracrystalline array, showing detail of the naked capsids containing an electron-dense core. Scale bar=500 nm.

Fig. 5.. Histopathology images of C. edule infected with BiIV1. (a) Infiltrating haemocytes within the connective tissues surrounding the digestive gland tubules (DG) were observed to possess basophilic inclusions (arrowheads) in the cytoplasm. Scale bar=50 µm. (b) Higher magnification of haemocytes with basophilic inclusions (black arrowheads) within the cytoplasm. Nuclei of the affected cells also showed marginated and condensed chromatin (white arrowheads). Scale bar=50 µm. Inset: higher magnification showing a basophilic inclusion (arrowhead) in the cytoplasm of a haemocyte. Scale bar=10 µm.