Twenty-Five New Viruses Associated with the Drosophilidae (Diptera)

Abstract

Drosophila melanogaster is an important laboratory model for studies of antiviral immunity in invertebrates, and Drosophila species provide a valuable system to study virus host range and host switching. Here, we use metagenomic RNA sequencing of about 1600 adult flies to discover 25 new RNA viruses associated with six different drosophilid hosts in the wild. We also provide a comprehensive listing of viruses previously reported from the Drosophilidae. The new viruses include Iflaviruses, Rhabdoviruses, Nodaviruses, and Reoviruses, and members of unclassified lineages distantly related to Negeviruses, Sobemoviruses, Poleroviruses, Flaviviridae, and Tombusviridae. Among these are close relatives of Drosophila X virus and Flock House virus, which we find in association with wild Drosophila immigrans. These two viruses are widely used in experimental studies but have not been previously reported to naturally infect Drosophila. Although we detect no new DNA viruses, in D. immigrans and Drosophila obscura, we identify sequences very closely related to Armadillidium vulgare iridescent virus (Invertebrate iridescent virus 31), bringing the total number of DNA viruses found in the Drosophilidae to three.

Keywords: Drosophila; Drosophila X virus; Flock House virus; metagenomics; transcriptome; virus.

Figure 1

Viruses related to well-studied clades. Mid-point rooted maximum-likelihood phylogenetic trees for the viruses reported here, inferred using polymerase protein sequences. Notes: The gray scale bars represent 0.5 amino acid substitutions per site. In each tree, viruses reported from Drosophilidae are labeled in red, viruses from other taxa are labeled in black, and unannotated virus-like sequences from publicly available transcriptome datasets are labeled in blue. Viruses newly reported here are underlined, and Drosophila species abbreviations are given for the reference sequence (Dimm, D. immigrans; Dobs, D. obscura; Dsub, D. subobscura; Dsus, D. subsilvestris; Dtri, D. tristis; Sdef, S. deflexa). Tree A: viruses near to the Dicistroviridae (Picornavirales); B: putative Cripaviruses (Dicistroviridae, Picornavirales – the corresponding tree in Supplementary File 4 additionally includes Aparaviruses); C: Nodaviruses; D: Birnaviruses; E: unclassified members of the Rhabdoviridae that form the sister clade to the Cytorhabdoviruses and the Nucleorhabdoviruses; and F: Reoviridae. Alignments are provided in Supplementary File 3, and clade support values and sequence accession identifiers are provided in Supplementary File 4.

Figure 2

Viruses not closely related to well-studied clades. Notes: See Figure 1 for a key to the colors and abbreviations. Tree A: unclassified Picornavirales; B: unclassified clade of basally branching Flavi-like viruses; C: an unclassified clade that branches basally to Poleroviruses and Sobemoviruses; C: Nodaviruses; D: Iflaviruses, including a new clade that falls within (or close to) the Iflaviruses; E: two unclassified clades related to the Tombusviridae; and F: two unclassified clades related to the Negeviruses and the Virgaviridae. Alignments are provided in Supplementary File 3, and clade support values and sequence accession identifiers are provided in Supplementary File 4.

Figure 3

Virus read numbers (relative to host COI, normalized for length). Notes: A heat map showing the relative number of high-quality (80 nt) forward reads from each library that map to each of the Drosophila viruses. Read numbers are normalized by target sequence length and by the number of reads mapping to a fragment of the host COI gene (so that a value of 1 implies equal read numbers per unit length of the virus and the host cytochrome oxidase 1). Rows and columns are clustered by the similarity in read frequency on a log scale. Note that some viruses may be sufficiently similar for a small proportion of reads to cross-map and that a small level of cross-contamination between fly species means that the data presented here cannot be used to confidently infer host specificity.