Viral Outbreak in Corals Associated with an In Situ Bleaching Event: Atypical Herpes-Like Viruses and a New Megavirus Infecting Symbiodinium

Abstract

Previous studies of coral viruses have employed either microscopy or metagenomics, but few have attempted to comprehensively link the presence of a virus-like particle (VLP) to a genomic sequence. We conducted transmission electron microscopy imaging and virome analysis in tandem to characterize the most conspicuous viral types found within the dominant Pacific reef-building coral genus Acropora. Collections for this study inadvertently captured what we interpret as a natural outbreak of viral infection driven by aerial exposure of the reef flat coincident with heavy rainfall and concomitant mass bleaching. All experimental corals in this study had high titers of viral particles. Three of the dominant VLPs identified were observed in all tissue layers and budding out from the epidermis, including viruses that were ∼70, ∼120, and ∼150 nm in diameter; these VLPs all contained electron dense cores. These morphological traits are reminiscent of retroviruses, herpesviruses, and nucleocytoplasmic large DNA viruses (NCLDVs), respectively. Some 300-500 nm megavirus-like VLPs also were observed within and associated with dinoflagellate algal endosymbiont (Symbiodinium) cells. Abundant sequence similarities to a gammaretrovirus, herpesviruses, and members of the NCLDVs, based on a virome generated from five Acropora aspera colonies, corroborated these morphology-based identifications. Additionally sequence similarities to two diagnostic genes, a MutS and (based on re-annotation of sequences from another study) a DNA polymerase B gene, most closely resembled Pyramimonas orientalis virus, demonstrating the association of a cosmopolitan megavirus with Symbiodinium. We also identified several other virus-like particles in host tissues, along with sequences phylogenetically similar to circoviruses, phages, and filamentous viruses. This study suggests that viral outbreaks may be a common but previously undocumented component of natural bleaching events, particularly following repeated episodes of multiple environmental stressors.

Keywords: herpesvirus; megavirus; nucleocytoplasmic large DNA virus (NCLDV); tropical coral reef; virome; virus-like particle (VLP).

FIGURE 1

Map of Heron Island tidal flat (Great Barrier Reef, Australia) indicating the location from which experimental coral colonies were collected (X). Photograph of tidal flat exemplifies the partial aerial exposure and associated patchy bleaching that many corals experienced in March 2011, prior to and in conjunction with the collection of experimental coral colonies.

FIGURE 2

Summary time line of conditions at the time of collection and observed bleaching at Heron Island. Upper composite graph generally indicates (from top to bottom) tide, temperature, and rain intensity. Tide is based on tide tables from the region (black line), with reef flat water depth (green line) as measured at the 1.1 m mooring present on the reef flat. Temperature is depicted as reef flat water depth (dark red line, measured at the 1.1 m water depth mooring), 8 m temperature (light green line, measured at the 7.9 m mooring), and air temperature (orange line). Key aspects of the temperature and tidal cycle (time points A, B and C in upper composite graph), the timing of coral collections (vertical dashed gray lines) and the authors’ first observation of mass bleaching on the reef flat (vertical red line) are also indicated. Lower right graph is a subset of information from the upper composite graph, highlighting the overlap between low tides on the reef flat (green line, measured at the 1.1 m water depth mooring), the tidal height (black line), and rainfall intensity (blue line). Air temperature and rain intensity were obtained from the local weather station on Heron Island. Lower left graph summarizes the mean temperature (±1 SD) and mean maximum temperature (±1 SD) and maximum temperature recorded at the reef flat (1.1 m water depth mooring) for the years 2008–2015. This figure is based on data provided by the Australian Institute of Marine Science.

FIGURE 3

Relative percentage of viral Families found in a single control Acropora aspera coral virome (generated from five coral fragments) using the best tBLASTx similarities to assembled contigs. Bar heights indicate the relative percentages of best similarities to viral Families (eukaryotic viruses) or categories of phages. Colors of bars distinguish phages (dark blue and green) from eukaryotic viruses, and genome type within the eukaryotic viruses (red = dsDNA, purple = ssDNA, light blue = RNA).

FIGURE 4

Representative examples of the acroporid atypical herpes-like virus from Acropora aspera coral fragments. These abundant herpes-like VLPs are comprised of enveloped, icosahedral (non-tailed) capsids ranging from 120 to 150 nm in diameter and contain electron dense cores. The VLPs in (F) are within a cellular vacuole; this is characteristic of herpesviruses. (B) is an enlargement of (A); (D) is an enlargement of (E); (H) is an enlargement of (G). (A,B) and (G–I) are images of the control saline-injected coral treatment fragments used to generate the viral metagenome. Arrows in (A–G) indicate general examples of atypical herpes-like viral particles. Asterisks indicate bacterial cells. Scale bars are 100 nm, unless otherwise noted.

FIGURE 5

Representative examples of a phycodnavirus-like nucleocytoplasmic large DNA virus (NCLDV) in Acropora aspera (A–E) and Acropora millepora(F–I) coral fragments. Asterisks indicate bacterial cells. Scale bars are as follows: (A,B,D–I) = 100 nm; (C) = 1 μm.

FIGURE 6

Representative examples of the giant Megaviridae-like NCLDV observed within Symbiodinium cells residing in Acropora aspera(A,B) and Acropora millepora(D) and near Symbiodinium cells in a probable A. aspera symbiosome (C). ch, chloroplast; py, pyrenoid; sy, Symbiodinium.

FIGURE 7

Representative examples of additional VLP diversity observed in Acropora aspera and Acropora millepora. In (A,B), arrows indicate star-shaped VLPs that are morphologically reminiscent of circovirus and nanovirus particles. (C,D) Depict retrovirus-like VLPs; the VLPs in (C) are within a cell vacuole. (E,F) Appear to be long filamentous VLPs within large ovoid viral factories. In (G), arrows indicate phage-like VLPs within bacterial cells (large, ovoid polygons) enclosed in peri-algal spaces of the host gastroderm. Scale bars are 200 nm, unless otherwise noted.