Revealing the determinants of virome composition is central to placing disease emergence in a broader evolutionary context. Fish are the most species-rich group of vertebrates and so provide an ideal model system to study the factors that shape virome compositions and their evolution. We characterised the viromes of 19 wild-caught species of marine fish using total RNA sequencing (meta-transcriptomics) combined with analyses of sequence and protein structural homology to identify divergent viruses that often evade characterisation. From this, we identified 25 new vertebrate-associated viruses and a further 22 viruses likely associated with fish diet or their microbiomes. The vertebrate-associated viruses identified here included the first fish virus in the Matonaviridae (single-strand, negative-sense RNA virus). Other viruses fell within the Astroviridae, Picornaviridae, Arenaviridae, Reoviridae, Hepadnaviridae, Paramyxoviridae, Rhabdoviridae, Hantaviridae, Filoviridae and Flaviviridae and were sometimes phylogenetically distinct from known fish viruses. We also show how key metrics of virome composition—viral richness, abundance and diversity—can be analysed along with host ecological and biological factors as a means to understand virus ecology. Accordingly, these data suggest that that the vertebrate-associated viromes of the fish sampled here are predominantly shaped by the phylogenetic history (i.e. taxonomic order) of their hosts, along with several biological factors including water temperature, habitat depth, community diversity and swimming behaviour. No such correlations were found for viruses associated with porifera, molluscs, arthropods, fungi and algae, that are unlikely to replicate in fish hosts. Overall, these data indicate that fish harbour particularly large and complex viromes and the vast majority of fish viromes are undescribed.

The Great Barrier Reef (GBR) – the largest coral reef ecosystem in the world – supports over 1200 fish species with some of the highest population densities and diversities observed in vertebrates, offering a high potential for virus transmission among species. As such, the GBR represents an exceptional natural ecosystem to determine the impact of host community diversity on virus evolution and emergence. In recent decades the GBR has also experienced significant threats of extinction, making it one of the most vulnerable ecosystems on the planet. Despite the global importance of the GBR, our understanding of virus diversity and connectivity in tropical reef fishes remains poor. Here, we employed metatranscriptomic sequencing to reveal the viromes of 61 reef fish species. This identified transcripts representing 132 putative viral sequences, 38 of which exhibited strong phylogenetic relationships to known vertebrate-associated viral genera, including a novel Santee-Cooper ranavirus (Iridoviridae). We found little evidence for virus transmission between fish species living within a very restricted geographical space – a 100 m2 coral reef ecosystem – suggesting that there might be important host barriers to successful cross-species transmission despite regular exposure. We also identified differences in virome composition among reef fish families, such that cryptobenthic reef fishes – characterized by small body sizes and short life-spans – exhibited greater virome richness compared to large reef fishes. This study suggests that there are important barriers to cross-species virus transmission, and that successful emergence in a reef fish community likely requires active host adaptation, even among closely related host species.

The flavivirids (family Flaviviridae) are a group of positive-sense RNA viruses that include well-documented agents of human disease. Despite their importance and ubiquity, the time-scale of flavivirid evolution is uncertain. An ancient origin, spanning millions of years, is supported by their presence in both vertebrates and invertebrates and by the identification of a flavivirus-derived endogenous viral element in the peach blossom jellyfish genome (Craspedacusta Sowerby, phylum Cnidaria), implying that the flaviviruses arose early in the evolution of the Metazoa. To date, however, no exogenous flavivirid sequences have been identified in these hosts. To help resolve the antiquity of the Flaviviridae, we mined publicly available transcriptome data across the Metazoa. From this, we expanded the diversity within the family through the identification of 32 novel viral sequences and extended the host range of the pestiviruses to include amphibians, reptiles, and ray-finned fish. Through cophylogenetic analysis we found cross-species transmission to be the predominate macroevolutionary event across the non-vectored flavivirid genera (median, 68%), including a cross-species transmission event between bats and rodents, although long-term virus-host co-divergence was still a regular occurrence (median, 23%). Notably, we discovered flavivirus-like sequences in basal metazoan species, including the first associated with Cnidaria. This sequence formed a basal lineage to the genus Flavivirus and was closer to arthropod and crustacean flaviviruses than those in the tamanavirus group that include a variety of invertebrate and vertebrate viruses. Combined, these data attest an ancient origin of the flaviviruses, likely close to the emergence of the metazoans 750–800 million years ago.

Biological invasions are among the biggest threats to freshwater biodiversity. This is increasingly relevant in the Murray-Darling Basin, Australia, particularly since the introduction of the common carp (Cyprinus carpio). This invasive species now occupies up to 90% of fish biomass, with hugely detrimental impacts on native fauna and flora. To address the ongoing impacts of carp, cyprinid herpesvirus 3 (CyHV-3) has been proposed as a potentially effective biological control agent. Crucially, however, it is unknown whether CyHV-3 and other cyprinid herpesviruses already exist in the Murray-Darling. Further, little is known about those viruses that naturally occur in wild freshwater fauna, and the frequency with which these viruses jump species boundaries. To document the evolution and diversity of freshwater fish viromes and better understand the ecological context to the proposed introduction of CyHV-3, we performed a meta-transcriptomic viral survey of invasive and native fish across the Murray-Darling Basin, covering over 2,200 km of the river system. Across a total of 36 RNA libraries representing 10 species, we failed to detect CyHV-3 nor any closely related viruses. Rather, meta-transcriptomic analysis identified 18 vertebrate-associated viruses that could be assigned to the Arenaviridae, Astroviridae, Bornaviridae, Caliciviridae, Coronaviridae, Chuviridae, Flaviviridae, Hantaviridae, Hepeviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae and Rhabdoviridae families, and a further 27 that were deemed to be associated with non-vertebrate hosts. Notably, we revealed a marked lack of viruses that are shared among invasive and native fish sampled here, suggesting that there is little virus transmission from common carp to native fish species, despite co-existing for over 50 years. Overall, this study provides the first data on the viruses naturally circulating in a major river system and supports the notion that fish harbour a large diversity of viruses with often deep evolutionary histories.