Generating viral metagenomes from the coral holobiont

Abstract

† These authors have contributed equally to this work.Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available) the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

Comparative Metagenomics of Viral Assemblages Inhabiting Four Phyla of Marine Invertebrates

“…This is similar to the results of previous studies that utilized similar viral genome isolation protocols, and likely results from DNA released from host, bacterial, and/or archaeal cells during the tissue homogenization process (Hewson et al, 2011a(Hewson et al, , 2013Gudenkauf et al, 2014). The low number of viral annotations may be a result of lack of representative viral genomes in public databases (Weynberg et al, 2014). The wide range of contig sequences could have resulted from differences in host tissue mass, amounts of mucosal epithelia from which viruses were extracted, or from differences in viral load.…”

“…This would indicate that NPV's detected in the 2011 Hewson et al study were most likely from amphipod hosts. Iridoviruses have been found within insects, mollusks, crustacean, and cnidarians in previous studies (Williams, 2008;Weynberg et al, 2014), but were detected in Demasterias imbricata, P. helianthoides (second diseased sample from the Seattle Aquarium), E. troscheli (second diseased sample from Cates Park Reef), and GenomePlex-prepared P. chilensis samples from Chile. They may have enveloped or non-enveloped icosahedral capsids (depending on if they resulted from budding or release via lysis) and contain a linear genome of 140-303 kb that encodes a highly variable number of genes (Darai, 1989).…”

“…This technique may be used as viral surveillance (Ng, 2010), or as a starting point for further investigation into microbe-microbe interactions, microbe-host interactions, pathogenesis, and disease diagnosis. The lack of representative viral genomes in public databases may decrease confidence in metaviromes annotation (Weynberg et al, 2014). However, metaviromics provides a strong starting point for future study of viral-host relationships.…”

Metatranscriptome analysis of the reef-building coral Orbicella faveolata indicates holobiont response to coral disease

“…Alternatively, GAPR-1 might also be one of the genes that viruses manipulate to evade lysosomal elimination. To unequivocally establish a link between WPD and viruses, future studies should include analyzing samples across a time series, potentially in combination with a viral enrichment approach (Soffer et al, 2014;Weynberg et al, 2014), to characterize viral communities associated with the coral holobiont before, during, and after a WPD outbreak. The reduced expression of a C-type lectin that recognizes bacterial pathogens and Symbiodinium (Vidal-Dupiol et al, 2009) detected in WPD affected corals, as well as a microbiome shift to opportunistic/pathogenic taxa and functional diversification (Table 2) suggest weakened antibacterial defense and possible symbiont loss.…”

Novel ssDNA Viruses Detected in the Virome of Bleached, Habitat-Forming Kelp Ecklonia radiata

“…Alternative methods, such as random priming-mediated sequence-independent single-primer amplification (RP-SISPA), could have been used, but this method can cause an overrepresentation in the datasets of viruses with large genomes (Adriaenssens et al, 2017). This bias away from small genomes has been noted by other authors (Karlsson et al, 2013;Weynberg et al, 2014).…”