Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here we report fourteen DNA viruses detected in a metagenomic analysis of approximately 6500 pool-sequenced Drosophila, sampled from 47 European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a dsRNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to 12 segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2% or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly-available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D. melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described in association with the Drosophilidae. This has limited the opportunity to use natural host-pathogen combinations in experimental studies, and may have biased our understanding of the Drosophila virome. Here we describe fourteen DNA viruses detectable by metagenomic analysis of 6.5 thousand pool-sequenced Drosophila, sampled from 47 European locations between 2014 and 2016. These include three new Nudiviruses, a new and divergent Entomopox virus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of Galbut virus, an RNA Partitivirus, segregating at very low frequency. Remarkably, we show that Vesanto virus, a small DNA virus previously described as a Bidnavirus, may be composed of up to 12 segments and represents a new lineage of segmented DNA viruses. Only two of the DNA viruses, Kallithea virus (Nudiviridae) and Vesanto virus (Bidna-virus like) are common, being found in 2% or more of wild flies. The other viruses are rare, with many likely to be represented by a single infected fly in the collection. We find that virus prevalence in Europe reflects that seen in publicly-available datasets, with Kallithea virus and Vesanto virus being commonly detectable in data from wild-caught flies and large population cages, and the others being rare or absent. These analyses suggest that DNA viruses are generally rarer than RNA viruses in D. melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redress the earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

The cross-species transmission (CST) of pathogens can have dramatic consequences, as highlighted by recent disease emergence events affecting human, animal and plant health. Understanding the ecological and evolutionary factors that increase the likelihood of disease agents infecting and establishing in a novel host is therefore an important research area. Previous work across different pathogens, including rabies virus (RABV), found that increased evolutionary distance between hosts reduces the frequency of cross-species transmission and of permanent host shifts. However, whether this effect of host relatedness still holds for transmission among recently diverged hosts is not well understood. We aimed to ask if high host relatedness can still increase the probability of a host shift between more recently diverged hosts, and the importance of this effect relative to ecological predictors. We first addressed this question by quantifying the CST frequency of RABV between North American bat species within the genus Myotis, using a multi-decade data set containing 128 nucleoprotein (N) RABV sequences from ten host species. We compared RABV CST frequency within Myotis to the rates of CST between nine genera of North American bat species. We then examined whether host relatedness or host range overlap better explains the frequency of CST seen between Myotis species. We found that at the within genus scale, host range overlap, rather than host relatedness best explains the frequency of CST events. Moreover, we found evidence of CST occurring among a higher proportion of species, and CST more frequently resulting in sustained transmission in the novel host in the Myotis dataset compared to the multi-genus dataset. Our results suggest that among recently diverged species, the ability to infect a novel host is no longer restricted by physiological barriers but instead is limited by physical contact. Our results improve predictions of where future CST events for RABV might occur and clarify the relationship between host divergence and pathogen emergence.

Mesoamerican amphibian declines in apparently pristine and protected habitats have been severe, especially at elevations above 500 m sea level and have been linked to emerging diseases and a changing climate. The Craugastor punctariolus species series of direct developing frogs is endemic to the region and used to be comprised of 33 species, seven of which have known populations at present. One of these, C. ranoides, endemic to southern Nicaragua and Costa Rica, was historically found in cloud forest sites of Area de Conservaci on Guanacaste (ACG) in north-west Costa Rica and extended into dry forest sites 20 km distant. Here, C. ranoides declined and disappeared from high elevation sites between the mid-1980s and early 1990s, but populations persisted in the lowland dry forest. We compared the genetic richness and ranavirus infection status of C. ranoides from extant dry forest populations to historic museum specimens of now extinct ACG cloud forest populations using DNA sequence diversity at two mitochondrial loci and molecular screening for ranavirus. Extant dry forest populations of C. ranoides formed a monophyletic group which included historic specimens sampled at cloud forest sites. However, the extirpated ACG cloud forest population contained additional diversity: samples formed a divergent clade with unknown spatial distribution. Ranavirus was detected in both current and museum samples of C. ranoides and sequences from a 267-nucleotide region of the major capsid protein gene shared 100% sequence identity with one another and with Frog virus 3. Our findings document cryptic diversity within an endangered species that has demonstrated no recovery in cloud forests and raises questions about Ranavirus and its potential link to the amphibian declines in this system. The presence of the same C. ranoides clade within present day and historical samples suggests a potential for effective translocation and repopulation of extirpated cloud forest populations.