Extensive Diversity of RNA Viruses in Australian Ticks

Abstract

Understanding the microbiome of ticks in Australia is of considerable interest given the ongoing debate over whether Lyme disease and its causative agent, the bacterium Borrelia burgdorferisensu lato, are present in Australia. The diversity of bacteria infecting Australian ticks has been studied using both culture- and metagenomics-based techniques. However, little is known about the virome of Australian ticks, including whether this includes viruses with the potential to infect mammals. We used a meta-transcriptomics approach to reveal the diversity and evolution of viruses from Australian ticks collected from two locations on the central east coast of Australia, including metropolitan Sydney. From this we identified 19 novel RNA viruses belonging to 12 families, as well as 1 previously described RNA virus. The majority of these viruses were related to arthropod-associated viruses, suggesting that they do not utilize mammalian hosts. However, two novel viruses discovered in ticks feeding on bandicoot marsupials clustered closely within the mammal-associated hepacivirus and pestivirus groups (family Flaviviridae). Another bandicoot tick yielded a novel coltivirus (family Reoviridae), a group of largely tick-associated viruses containing the known human pathogen Colorado tick fever virus and its relative, Eyach virus. Importantly, our transcriptomic data provided no evidence for the presence of B. burgdorferisensu lato in any tick sample, providing further evidence against the presence of Lyme disease in Australia. In sum, this study reveals that Australian ticks harbor a diverse virome, including some viruses that merit additional screening in the context of emerging infectious disease.IMPORTANCE Each year a growing number of individuals along the east coast of Australia experience debilitating disease following tick bites. As there is no evidence for the presence of the causative agent of Lyme disease, Borrelia burgdorferisensu lato, in Australian ticks, the etiological basis of this disease syndrome remains controversial. To characterize the viruses associated with Australian ticks, particularly those that might be associated with mammalian infection, we performed unbiased RNA sequencing on 146 ticks collected across two locations along the coast of New South Wales, Australia. This revealed 19 novel RNA viruses from a diverse set of families. Notably, three of these viruses clustered with known mammalian viruses, including a novel coltivirus that was related to the human pathogen Colorado tick fever virus.

Keywords: Ixodes holocyclus; RNA virus; coltivirus; marsupial; phylogeny; ticks; virome.

FIG 1

(A) Map of Australia showing the location of New South Wales, Australia, where sampling took place. (B) Locations of tick sampling sites, marked by solid black dots on a map of New South Wales. (C) Maximum likelihood phylogeny of the mitochondrial cytochrome c oxidase subunit I (COX1) gene from each pooled library, with the sampling species (and unfed ticks) and location indicated. Reference COX1 sequences from a variety of tick species are shown in red. The scale bar indicates the number of nucleotide substitutions per site. The tree was midpoint rooted for clarity only.

FIG 2

(A) Percentage of reads aligning to the tick mitochondrial COX1 reference sequence. (B) Percentage of reads aligning to RNA virus genomes in each library. The library composition is described in Table 1.

FIG 3

Abundance of viruses from each family or order, shown as the percentage of the total number of viral reads in each library.

FIG 4

Phylogenetic relationships and genomic structure of the +ssRNA viruses sampled in this study. All trees are scaled according to the number of amino acid substitutions per site. The trees are midpoint rooted for clarity only, and bootstrap values (>70%) are shown. Viruses determined here are shown in red. The symbols denote the vertebrate host that the tick was taken from or whether the virus was sampled from an unfed tick. Virus lineages previously shown to infect mammals or mammalian cells are shown in blue. Genome diagrams provide information on the length of each genomic segment, the number of ORFs, and predicted conserved protein structures. No diagrams are provided for viruses for which no full genomes were identified.

FIG 5

Phylogenetic relationships and genomic structure of the dsRNA viruses sampled in this study. All trees are scaled according to the number of amino acid substitutions per site. The trees were midpoint rooted for clarity only, and bootstrap values (>70%) are shown. Viruses determined here are shown in red. The symbols denote the vertebrate host that the tick was taken from or whether the virus was sampled from an unfed tick. Virus lineages previously shown to infect mammals or mammalian cells are shown in blue. Genome diagrams provide information on the length of each genomic segment, the number of predicted ORFs, and predicted conserved protein structures.

FIG 6

Phylogenetic relationships and genomic structure of the −ssRNA viruses sampled in this study. All trees are scaled according to the number of amino acid substitutions per site. The trees are midpoint rooted for clarity, and bootstrap values (>70%) are shown. Viruses determined here are shown in red. The symbols denote the vertebrate host that the tick was taken from or whether the virus was sampled from an unfed tick. Virus lineages previously shown to infect mammals or mammalian cells are shown in blue. Genome diagrams provide information on the length of each genomic segment, the number of predicted ORFs, and predicted conserved protein structures.