Archives of Virology (2021) 166:1843–1852 https://doi.org/10.1007/s00705-021-05060-8 ORIGINAL ARTICLE Sequence diversity and evolution of a group of ifaviruses associated with ticks Romain Daveu1,2 · Caroline Hervet1 · Louane Sigrist1 · Davide Sassera2 · Aaron Jex3,4 · Karine Labadie5 · Jean‑Marc Aury6 · Olivier Plantard1 · Claude Rispe1 Received: 9 November 2020 / Accepted: 15 February 2021 / Published online: 19 April 2021 © The Author(s) 2021 Abstract We studied a group of tick-associated viruses with characteristics of members of the family Ifaviridae, a family of viruses frequently found in arthropods. Our aim was to gain insight into the evolutionary dynamics of this group of viruses, which may be linked to the biology of ticks. We explored assembled RNA-Seq data sets for diferent species of ticks. We identi- fed members of fve diferent ifavirus species, four of them novel, and discovered nine new genome sequences, including variants. Five variants represented a virus species associated with Ixodes ricinus. Unexpectedly, a sequence found in the Ixodes scapularis cell line ISE6 was nearly identical to the sequences of I. ricinus variants, suggesting a contamination of this cell line by I. ricinus material. Analysing patterns of substitutions between these variants, we detected a strong excess of synonymous mutations, suggesting evolution under strong positive selection. The phylogenies of the viruses and of their tick hosts were not congruent, suggesting recurrent host changes across tick genera during their evolution. Overall, our work constitutes a step in the understanding of the interactions between this family of viruses and ticks. Introduction RNA viruses has rapidly increased in recent years, and they appear to be far more prevalent and diverse than previously Ticks are blood-feeding parasites, which makes of them a expected, especially in invertebrates [1, 2]. High-throughput "hub" for a number of microorganisms (bacterial pathogens transcriptome sequencing (or RNA-Seq) has proven to be and symbionts, viruses, protozoa) that potentially interact the tool of choice for characterizing the genomes of these with ticks and their vertebrate hosts. The discovery of new viruses in many types of arthropods [2, 3], including ticks [4, 5], as this technique does not require known sequences to be targeted, while allowing the retrieval of contigs that Handling Editor: Simona Abba. * Claude Rispe Olivier Plantard [email protected] [email protected] Romain Daveu 1 INRAE, Oniris, BIOEPAR, Nantes, France [email protected] 2 Department of Biology and Biotechnology “L. Spallanzani”, Caroline Hervet University of Pavia, Pavia, Italy [email protected] 3 Population Health and Immunity Division, Walter and Eliza Louane Sigrist Hall Institute of Medical Research, Parkville, VIC 3052, [email protected] Australia Davide Sassera 4 Faculty of Veterinary and Agricultural Sciences, The [email protected] University of Melbourne, Parkville, VIC 3010, Australia Aaron Jex 5 Genoscope, Institut de biologie François-Jacob, [email protected] Commissariat à l’Energie Atomique (CEA), Université Karine Labadie Paris-Saclay, Evry, France [email protected] 6 Génomique Métabolique, Genoscope, Institut François Jean-Marc Aury Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, [email protected] 91057 Evry, France Vol.:(0123456789)1 3 1844 R. Daveu et al. may contain full viral genome sequences, given a sufcient uncultivated virus genomes (UViGs). Genomes were coverage and sufcient prevalence of the virus in the ana- defned as representing novel species if their sequence lysed samples. The Ifaviridae are a family of RNA viruses identity to members of existing species was less than 90%, within the order Picornavirales. They are non-enveloped according to the species delineation criteria of the Inter- and have a positive-sense, single-stranded non-segmented national Committee on Taxonomy of Viruses (ICTV) for genome of ~9-11 kb [6]. This family of viruses has been ifaviruses (https:// talk. ictvo nline. org/ ictv- repor ts/ ictv_ shown to have a privileged association with insects, being online_ report/ posit ive- sense- rna- virus es/w/ ifav iridae). detected in a growing number of species belonging to several We also performed a BLASTp search on the nr database, orders. Ifavirus infections can range from asymptomatic to selecting all of the matches described as being associated severe, causing developmental anomalies (e.g., deformed with ticks and adding them to our data set. This approach wing virus in honey bees) or death [6]. Ifaviruses have also ensured that our study included all of the tick-associated been detected in parasitic mites (subphylum Chelicerata) ifavirus genome sequences published to date (as of Sep- [7]. Recently, genomes of viruses belonging to the family tember 2020). Ifaviridae have been detected in pools of tick species [2], in an endemic Australian tick species (Ixodes holocyclus ifavirus [IhIV]) [8], in a tick species associated with marine Phylogeny methods birds, Ixodes uriae [9, 10] (Gerbovitch virus), and in the ISE6 tick cell line (Ixodes scapularis ifavirus [ISIV]) [11]. Based on a recent phylogenetic study of ifaviruses [14], we Overall, members of the family Ifaviridae are among the included two outgroups, one associated with the mite Tetra- most common viruses that have been identifed in diferent nychus truncatus (representing the closest outgroup to tick- species of ticks, although many aspects of their biology are associated ifaviruses) and the other associated with Apis still unknown, including their mode of transmission, their mellifera (deformed wing virus [DWV]). We aligned amino efect on the ftness of the tick host, and the phylogenetic acid sequences of all ifavirus polyprotein sequences using relationships among members of this family. For this reason, MUSCLE in MEGA X [15]. This alignment was fltered we decided to explore the many transcriptome data sets that using Gblocks [16] to exclude poorly aligned regions and have been published for diferent tick species, searching for gaps. The fltered alignment comprised 1,149 amino acid new sequences of members of the family Ifaviridae. We positions. A phylogenetic ML tree was constructed using IQ- identifed nine new genome sequences representing fve dif- TREE [17]. The best model of substitution was determined ferent species of tick-associated ifaviruses, including several using Model Finder [18], and branch support was assessed variants of the same virus species in Ixodes ricinus, and using 1000 ultrafast bootstrap replicates [19]. A graphical studied the evolution and phylogenetic relationships of this representation of the consensus ML tree was made using group. ITOL [20]. Materials and methods Test of congruence between host and virus phylogenies Search for ifavirus sequences To test the congruence between virus and host phylog- We searched for sequences matching ifaviruses in pub- enies, we used the cophylogeny testing tool Jane 4 [21]. lic tick transcriptome assemblies (available from TSA, The host phylogeny was derived from a previous transcrip- GenBank, as of August 2020). For each transcriptome tome-based phylogenetic study of hard ticks, using a large assembly, we searched for matches using tblastn [12], number of nuclear markers [13]. Three ifavirus genomes, using the polyprotein sequence of ISIV (accession number obtained from pools of diferent species of ticks, could not BBD75427) as a query and RNA-Seq contigs (nucleotide be included in this analysis because it was impossible to sequences) as targets. Of note, several of these transcrip- assign a host species to these ifaviruses. We also collapsed tomes were produced by our research group for a phylo- branches containing virus sequences that were nearly identi- genetic study of hard ticks [13] or for expression profl- cal in the virus phylogeny, since they can be considered to ing studies of I. ricinus (BioProject accession numbers represent the same species. This resulted not only in elimi- PRJEB40724 and PRJNA662253). We used low-strin- nating two ifavirus sequences associated with I. holocyclus gency matching criteria (e-value threshold 1-e3) to ensure but also another group of six sequences, fve of which were that even relatively distantly related sequences could be found in I. ricinus and one of which was found in a cell found. Since new genome sequences identifed in our study line of I. scapularis (see “Results”). The name given to this were retrieved from transcriptome data, they represent group was IricIV-ISIV. 1 3 Ifaviruses associated with ticks 1845 Estimation of genetic distances and evolutionary frst two of these viruses, the retrieved sequences were rates incomplete (5’ partial), and the sequenced region of the ORF comprised 2,437, and 1,945 amino acids, represent- We estimated genetic distances and evolutionary rates within ing ~81% and ~65% of the complete genome, respec- two ensembles of sequences, one including ISIV and I. rici- tively. In the case of HydromIV, the match with the ISIV nus variants and the other including two variants associated sequence was in two frames, suggesting a frameshift. This with I. holocyclus, that grouped closely in the phylogenetic frameshift could be authentic, especially if this sequence analysis. We frst estimated genetic distances (number of corresponds to an endogenous viral element (EVE), given substitutions per site) at both the nucleotide level and the that EVEs do not necessarily