Characterisation of the virome of Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae) , a vector of bluetongue virus in Australia

Abstract

Culicoides spp., a common biting midge genus, are haematophagous insects that can transmit pathogens to humans and other animals. Some species transmit arboviruses, including bluetongue virus, epizootic haemorrhagic disease virus, African horse sickness virus and Schmallenberg virus to vertebrates, which can be detrimental to livestock and wild animals. Culicoides spp. can also have a diversity of insect-specific viruses (ISVs) that can only be transmitted between insects and others related to known arboviruses. For Culicoides brevitarsis and other Culicoides spp. in Australia, the virome is largely unexplored. We used high-throughput sequencing to characterise the virome of C. brevitarsis collected from Casino, New South Wales, Australia. For virus detection, the total RNA was extracted from pools of C. brevitarsis followed by rRNA depletion and Illumina short-read-based RNA sequencing. The reads were quality-checked, filtered and assembled into contigs, compared with the non-redundant protein and conserved domain databases for viral detection and genome organisation, respectively. The phylogenetic analysis was used to further characterise the viruses. We detected new virus diversity including ten viruses belonging to eight different families with complete or near-complete coding regions. Seven of these were novel virus species belonging to the families: Chuviridae, Orthomyxoviridae, Peribunyaviridae, Qinviridae, Rhabdoviridae and Solemoviridae. In addition, the novel Peribunyaviridae virus should also be considered part of a new genus. Whilst most of the detected viruses grouped into families with viruses that can infect insects, animals or both, the novel species of Solemoviridae was closely related to an economically important plant pathogen, the sugarcane yellow leaf virus. Our quantitative PCR-based screening confirmed the absence of any Wolbachia endosymbiont within the collected samples. Furthermore, we detected fragments of three more virus families known to infect fungi and plants. The detection of potential arboviruses and ISVs in Culicoides spp. is important in understanding virus epidemiology.

Keywords: Culicoides; RNA virus; arbovirus; insect-specific virus; vector.

Fig. 1.. Genome organisation charts of assembled virus segments in this study. The protein domain positions of 14 assembled virus segments belonging to 10 viruses with complete or near-complete coding regions were predicted by comparing each assembled viral contig to the CDD. The unit of the X-axis is in bp. The abbreviations S1, S2 and S3 correspond to each segment where a virus was segmented. Incomplete coding regions are indicated by an asterisk.

Fig. 2.. Phylogenetic trees for Chuviridae and Qinviridae. Maximum likelihood phylogenetic trees based on MAFFT alignments of the conserved RNA-dependent RNA polymerase and RdRP gene region of (a) Chuviridae using the LG+F+I+G4 model and (b) Qinviridae using the Q.pfam+F+I+G4 model. The outgroup for (a) is Ebola virus and for (b) is Ourmia melon virus. The support values are presented at the node based on 1000 bootstraps. The scale bars represent the number of amino acid substitutions per site.

Fig. 3.. Phylogenetic trees for Rhabdoviridae, Peribunyaviridae and Orthomyxoviridae. Maximum likelihood phylogenetic trees based on MAFFT alignments of the conserved RNA-dependent RNA polymerase and RdRP gene region of (a) Rhabdoviridae using the Q.yeast+I+G4 model, (b) Peribunyaviridae using the Q.yeast+F+I+G4 model and (c) Orthomyxoviridae using the LG+I+G4 model. The outgroup for (a) is Nyaminini virus and for (b) and (c) is watermelon crinkle leaf-associated virus 2. The support values are presented at the node based on 1000 bootstraps. The scale bars represent the number of amino acid substitutions per site.

Fig. 4.. Phylogenetic trees for Solemoviridae. Maximum likelihood phylogenetic trees based on MAFFT alignments of the conserved RNA-dependent RNA polymerase and RdRP gene region of Solemoviridae using the LG+I+G4 model. The outgroup is the mushroom bacilliform virus. The support values are presented at the node based on 1000 bootstraps. The scale bars represent the number of amino acid substitutions per site.