Isolation and characterization of Nylanderia fulva virus 1, a positive-sense, single-stranded RNA virus infecting the tawny crazy ant, Nylanderia fulva

Abstract

We report the discovery of Nylanderia fulva virus 1 (NfV-1), the first virus identified and characterized from the ant, Nylanderia fulva. The NfV-1 genome (GenBank accession KX024775) is 10,881 nucleotides in length, encoding one large open reading frame (ORF). Helicase, protease, RNA-dependent RNA polymerase, and jelly-roll capsid protein domains were recognized within the polyprotein. Phylogenetic analysis placed NfV-1 in an unclassified clade of viruses. Electron microscopic examination of negatively stained samples revealed particles with icosahedral symmetry with a diameter of 28.7±1.1nm. The virus was detected by RT-PCR in larval, pupal, worker and queen developmental stages. However, the replicative strand of NfV-1 was only detected in larvae. Vertical transmission did not appear to occur, but horizontal transmission was facile. The inter-colonial field prevalence of NfV-1 was 52±35% with some local infections reaching 100%. NfV-1 was not detected in limited samples of other Nylanderia species or closely related ant species.

Keywords: Genome sequence; Insect picorna-like virus; Nylanderia fulva; Tawny crazy ant, RNA virus.

Graphical abstract

Fig. 1

(A) NfV-1 genome organization and method of acquisition. The upper blue arrows represent the cloning strategy for acquiring the NfV-1 genome. Contig 3776.C1 was used as template for initial 5′ and 3′ RACE reactions. Positions of picorna-like helicase (Hel), protease (Pro) and RNA-dependent RNA polymerase (RdRp) protein domains are shown within the polyprotein ORF. Predicted ovarian tumor (OTU), picorna/calici-like jelly-roll fold capsid protein (JR) and dsRNA binding protein (*) domains are also indicated. Potential virus protease cleavage sites are indicated with red triangles. The sequence upstream of Pro is likely to correspond to VPg. A putative sgRNA is indicated. Initiation at the first AUG (poor context) on the sgRNA would lead to translation of a short overlapping ORF while initiation at the second AUG (strong context) would lead to translation of the dsRBP domain and capsid proteins. VP1 (orange) and VP2 (red) are annotated based on analogy with the mapped structural proteins of SINV-3. (B) SINV-3 genome organization and potential cleavage sites. (C) Sequences at the potential cleavage sites in NfV-1 and SINV-3. (D) Comparison of the 5′ terminal sequences of the NfV-1 gRNA and putative sgRNA.

Fig. 2

Genome maps of APV, SINV-3, NfV-1, related viruses, and TSA sequences likely to represent additional related viruses. Nora virus, the next most closely related virus (by RdRp-based phylogeny), is also shown for comparison. Sequences assembled from Roche 454 sequence data are shown in pale green with breaks in the polyprotein ORF presumed to have resulted from frame-shift sequencing errors indicated. (The Sitobion TSA also derives from 454 sequencing but high similarity to APV permitted unambiguous removal of three frame-shift errors.) Likely partial sequences are also indicated by un-closed rectangles, although it is possible that additional sequences are incomplete and therefore that some ORFs may be 5′-truncated. Red lines in KFV indicate a 638-nt sequence duplication that may indicate a rearranged defective sequence. Experimentally mapped capsid proteins for APV (van der Wilk et al., 1997), KFV (Hartley et al., 2005), Nora virus (Ekström et al., 2011), and SINV-3 (Valles et al., 2014a) are indicated in colors other than blue and green. Ribosomal −1 frameshift sites are indicated. Protein domains identified by conserved characteristic motifs (Hel – superfamily III helicase; Pro – 3C-like protease; RdRp – superfamily I RdRp) (Koonin et al., 2008) or HHpred (* – dsRNA binding domain; Z – zinc-finger domain; JR – picorna/calici-like jelly-roll capsid domain), as well as the baculoviral inhibitor of apoptosis repeat domain (I) in KFV identified by Hartley et al. (2005), are indicated. The SINV-3 sgRNA and corresponding putative sgRNAs for APV and NfV-1 are indicated; however we expect that all these viruses produce a similarly located sgRNA for capsid protein expression.

Fig. 3

Phylogenetic tree for picorna-like viruses. RdRp amino acid sequences from picorna-like viruses were obtained from Koonin et al. (2008), combined with the equivalent regions from the 17 sequences in Supplementary Table 1, realigned with MUSCLE, and a Bayesian Markov chain Monte Carlo based phylogenetic tree produced. Posterior probabilities are indicated where p<1.00. The APV-like and SINV-3/NfV-1-like clades discussed herein are indicated with green and red ellipses, respectively. Within these two clades, posterior probabilities were >0.9 except for the placement of Nora virus within these two clades (p=0.64), the placement of the Liposcelis and Leptinotarsa TSAs within the SINV-3 clade (p=0.68), and the placement of the Eucyclops and Anurida TSAs within the APV clade (p=0.60). Abbreviations: AhV, Atkinsonella hypoxylon virus; ALSV, apple latent spherical virus; ANV, avian nephritis virus; APV, Acyrthosiphon pisum virus; BaYMV, barley yellow mosaic virus; BBWV-1, broad bean wilt virus 1; BDRC, Bryopsis cinicola chloroplast dsRNA replicon; BDRM, Bryopsis mitochondria-associated dsRNA; BWYV, beet western yellows virus; CHV, Cryphonectria parasitica hypovirus; CPMV, cowpea mosaic virus; CPV, Cryptosporidium parvum virus; CrPV, cricket paralysis virus; DCV, Drosophila C virus; DWV, deformed wing virus; EMCV, encephalomyocarditis virus; FCCV, Fragaria chiloensis cryptic virus; FCV, feline calicivirus; FGMV, Fusarium graminearum mycovirus; FHV, flock house virus; FMDV, foot-and-mouth disease virus; GFLV, grapevine fanleaf virus; GLV, Giardia lamblia virus; HaRNAV, Heterosigma akashiwo RNA virus; HAstV1, human astrovirus 1; HAV, hepatitis A virus; HcRNAV, Heterocapsa circularisquama RNA virus; HRV1A, human rhinovurus 1A; IFV, infectious flacherie virus; JP-A, marine RNA virus JP-A; JP-B, marine RNA virus JP-B; KFV, kelp fly virus; KiV, Kilifi virus; LRV, leishmania RNA virus 1-1; LTSV, lucerne transient streak virus; MBV, mushroom bacilliform virus; NfV-1, Nylanderia fulva virus 1; NoroV, norovirus; NoV, Nodamura virus; OAstV1, ovine astrovirus 1; OPV, Ophiostoma partitivirus 1; PEMV-1, pea enation mosaic virus 1; PLRV, potato leafroll virus; PnPV, Perina nuda picorna-like virus; PV, poliovirus; PYFV, parsnip yellow fleck virus; RAAV, rosy apple aphid virus; RasR1, Raphanus sativus dsRNA 1; RHDV, rabbit haemorrhagic disease virus; RsRNAV, Rhizosolenia setigera RNA virus; RTSV, rice tungro spherical virus; SBMV, southern bean mosaic virus; SCPMV, southern cowpea mosaic virus; ScV, Saccharomyces cerevisiae virus LA; SDV, satsuma dwarf virus; SINV-2, Solenopsis invicta virus 2; SINV-3, Solenopsis invicta virus 3; SJNNV, striped jack nervous necrosis virus; SmVA, Sclerophtora macrospora virus A; SmVB, Sclerophtora macrospora virus B; SPMMV, sweet potato mild mottle virus; SssRNAV, Schizochytrium single-stranded RNA virus; SV, Sapporo virus; TAstV1, turkey astrovirus 1; TEV, tobacco etch virus; ThV, Thika virus; TRSV, tobacco ringspot virus; TrV, Triatoma virus; TSV, Taura syndrome virus; TVV, Trichomonas vaginalis virus 1; WSMV, wheat streak mosaic virus.

Fig. 4

Electron micrograph of a negative stain of Nylanderia fulva virus 1 purified by CsCl isopycnic centrifugation. A group of intact virus particles demonstrating the icosahedral symmetry (arrow) and a surface with numerous small projections (spikes). Mean diameter of virus particles was 28.7±1.1 nm.

Fig. 5

Intra-colonial prevalence (upper) and quantity (lower) of Nylanderia fulva virus 1 in different developmental stages of the host, Nylanderia fulva. Colonies were pre-determined to be infected with NfV-1 and then individuals from different life stages were tested to determine the mean prevalence of the virus by RT-PCR. Individuals testing positive by RT-PCR were subsequently evaluated by QPCR.