Discovery of three RNA viruses using ant transcriptomic datasets
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Three novel RNA viruses, named Formica fusca virus 1 (GenBank accession no. MH477287), Lasius neglectus virus 2 (MH477288) and Myrmica scabrinodis virus 2 (MH477289), were discovered in ants collected in Cambridge, UK. The proposed virus names were given based on the hosts in which they were identified. The genome sequences were obtained using de novo transcriptome assembly of high-throughput RNA sequencing reads and confirmed by Sanger sequencing. Phylogenetic analysis showed that Formica fusca virus 1 grouped within the family Nyamiviridae, Lasius neglectus virus 2 grouped within the family Rhabdoviridae and Myrmica scabrinodis virus 2 belongs to the family Dicistroviridae. All three viruses are highly divergent from previously sequenced viruses.
Insect-infecting viruses comprise a large and diverse group, but their full diversity has as yet been poorly investigated . Further investigation may provide new insights into the long-term evolution and origin of viruses and may also reveal new tools for molecular biology research and biotechnology. Also, the host range of some of these viruses may make them suitable to use as biological control agents against invasive or pestiferous insect species.
We generated RNA-Seq libraries from ants sampled from various locations in Cambridge, UK . Sequencing data have been deposited in ArrayExpress (http://www.ebi.ac.uk/arrayexpress) under the accession number E-MTAB-5781. Trimmed RNA-Seq reads were assembled de novo into contigs using TRINITY v2.3.2 [3, 4], and assembled contigs were compared using BLASTX  against a database of all RNA virus protein sequences obtained from GenBank as described previously . Although we focused on genetically characterising three of the viral sequences with highest coverage, our analysis of Trinity assemblies revealed that further uncharacterised virus sequences were present in the RNA-Seq datasets. Primers were designed based on the three viral sequences, and overlapping fragments were amplified by PCR and sequenced by the Sanger method. Apart from the first 202 nt of FfusV-1 and the terminal 59 nt of MsaV-2, sequences of the entire genomes were obtained by Sanger sequencing; 100% nucleotide identity was confirmed between assembled and amplified sequences.
To determine the taxonomy of the three viruses, related sequences were identified within GenBank (5 Dec 2017, taxID = 10239 “viruses”), using the amino acid sequence of the entire ORF encoding the RNA-dependent RNA polymerase (RdRp) as the query in TBLASTN. Reference (RefSeq database) and selected non-reference (nr/nt database) sequences with > 25% coverage match were used for phylogenetic analysis. Amino acid sequences of the RdRp-encoding ORF were aligned using MUSCLE (v3.8.31) , and phylogenetic trees were inferred using MRBAYES v3.2.6 , with one million generations and default parameters. Phylogenetic trees were visualized with FIGTREE v1.4.3. To determine genome organisations, potential viral ORFs were identified using GETORF from the EMBOSS package , and each translated ORF was queried with HHpred  against the Pfam  and PDB  databases and identified based on homology to previously annotated proteins.
The sequence of Lasius neglectus virus 2 (LneV-2) comprises 12,041 nt. The 5′ leader and 3′ trailer sequences of the antigenome are of 115 and 174 nt, respectively, and share nine nucleotides of terminal complementarity. LneV-2 follows a typical Mononegavirales genome organisation, encoding proteins in the same order as FfusV-1, N (470 aa), P (433 aa), M (248 aa), G (506 aa) and L (2124 aa) (Fig. 1). As with FfusV-1, the proteins encoded by ORFs 2 and 3 were not confidently predicted by HHpred but inferred based on position in the genome. Genome coverage was obtained to a mean depth of 45 for the negative strand and 14 for the positive strand, again consistent with active virus replication. Phylogenetic analysis indicated that LneV-2 belongs in the family Rhabdoviridae but does not belong to any of the defined genera within the family; instead it falls within an unclassified cluster of arthropod-infecting rhabdoviruses. Based on the phylogenetic tree, LneV-2 is most closely related to Hubei rhabdo-like virus 1, Spodoptera frugiperda rhabdovirus and Wuhan ant virus (Fig. 2).
The sequence of Myrmica scabrinodis virus 2 (MsaV-2) comprises 10,663 nt. It contains two ORFs of 5553 nt and 4089 nt in length separated by an intergenic sequence of 196 nt and flanked by 5′ and 3′ UTRs of 630 and 199 nt plus the poly(A) tail, respectively (Fig. 1). HHpred analysis indicated that ORF 1 encodes the non-structural proteins helicase, protease and RdRp, whereas ORF 2 encodes three jelly-roll structural capsid proteins. Genome coverage was obtained to a mean depth of 262 for the positive strand and 0.2 for the negative strand. Importantly, the negative-strand reads – although sparse – were distributed throughout the genome, indicating the probable presence of a full-length negative strand. Phylogenetic analysis indicated that this virus belongs in the genus Triatovirus within the family Dicistroviridae of the order Picornavirales (Fig. 2). In dicistroviruses, the second ORF starts at a non-AUG initiation site due to the presence of an unusual intergenic region internal ribosomal entry site (IGR-IRES), which directs translation of the 3′ ORF via an initiator-Met-tRNA independent mechanism ; a typical IGR-IRES structure was predicted in MsaV-2 and used to infer the non-AUG start site of ORF 2.
This work was supported by a Wellcome Trust grant  and a European Research Council grant  to A.E.F.
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Conflict of interest
The authors declare that they have no conflict of interest.
The research reported here did not involve any studies on human subjects or vertebrate animals performed by any of the authors.
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