Background: Ticks are implicated as hosts to a wide range of animal and human pathogens. The full range of microbes harbored by ticks has not yet been fully explored. Methods: As part of a viral surveillance and discovery project in arthropods, we used unbiased high-throughput sequencing to examine viromes of ticks collected on Long Island, New York in 2013. Results: We detected and sequenced the complete genome of a novel rhabdovirus originating from a pool of Amblyomma americanum ticks. This virus, which we provisionally name Long Island tick rhabdovirus, is distantly related to Moussa virus from Africa. Conclusions: The Long Island tick rhabdovirus may represent a novel species within family Rhabdoviridae

AMQ King

CA Whitehouse

DM Stone

E Ghedin

G Palacios

HM Savage

IV Kuzmin

J Goddard

Komal Jain

M Labuda

Maria Sanchez Leon

N Vasilakis

O Poch

PL Quan

Rafal Tokarz

RC Kading

SR Telford

Stephen Sameroff

W Ian Lipkin

Virology Journal

PubMed

Crossref

Genome characterization of Long Island tick rhabdovirus, a new virus identified in Amblyomma americanum ticks

Tokarz, Rafal

Sameroff, Stephen Christopher

Leon, Maria

Jain, Komal

Lipkin, W. Ian

Columbia University Academic Commons

Tokarz et al. Virology Journal 2014, 11:26http://www.virologyj.com/content/11/1/26RESEARCH Open AccessGenome characterization of Long Island tickrhabdovirus, a new virus identified inAmblyomma americanum ticksRafal Tokarz*, Stephen Sameroff, Maria Sanchez Leon, Komal Jain and W Ian LipkinAbstractBackground: Ticks are implicated as hosts to a wide range of animal and human pathogens. The full range ofmicrobes harbored by ticks has not yet been fully explored.Methods: As part of a viral surveillance and discovery project in arthropods, we used unbiased high-throughputsequencing to examine viromes of ticks collected on Long Island, New York in 2013.Results: We detected and sequenced the complete genome of a novel rhabdovirus originating from a pool ofAmblyomma americanum ticks. This virus, which we provisionally name Long Island tick rhabdovirus, is distantlyrelated to Moussa virus from Africa.Conclusions: The Long Island tick rhabdovirus may represent a novel species within family Rhabdoviridae.Keywords: Ticks, Rhabdovirus, High-throughput sequencing, Amblyomma americanumBackgroundThe family Rhabdoviridae consists of a large group ofenveloped, single-stranded, negative sense RNA virusesthat infect a wide range of vertebrates, invertebrates,and plants [1]. Their genome typically consists of at leastfive open reading frames (ORFs) organized in a linearorder 3′-N-P-M-G-L-5′, and encode the viral nucleocap-sid (N), phosophoprotein (P), matrixprotein (M), glyco-protein (G) and RNA polymerase (L). In addition to thesegenes, many rhabdoviruses contain smaller ORFs thatencode additional accessory proteins, most withoutknown function. Currently, Rhabdoviridae consists ofnine named genera (Cytorhabdovirus, Ephemerovirus, Lys-savirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdo-virus, Sigmavirus, Tibrovirus, Vesiculovirus), althoughmany tentative rhabdoviruses still await taxonomic classi-fication [2].Arthropods are essential in transmission of many patho-genic rhabdoviruses. In the context of a program in viralsurveillance and discovery in arthropods, we examined* Correspondence: rt2249@cumc.columbia.eduCenter for Infection and Immunity, Mailman School of Public Health,Columbia University, 722 West 168th Street, Room 1701, New York, NY10032, USA© 2014 Tokarz et al.; licensee BioMed CentralCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.viromes of ticks collected in New York State and identi-fied a novel rhabdovirus associated with the lone star tick,Amblyomma americanum. We provisionally name thisvirus Long Island tick rhabdovirus.ResultsAnalysis of high-throughput sequencing (HTS) data byBLASTx revealed sequences with homology to all fiveprototypical rhabdovirus proteins. Homology searchesindicated these sequences were most similar to Moussavirus (MOUV) and therefore all were assembled toMOUV as a reference genome [3]. Amino acid analysisof the coding sequence suggested this virus likely repre-sented a novel rhabdovirus; hence, we tentatively namedit Long Island tick rhabdovirus (LITRV), after its geo-graphical location and host.GenomeThe complete genome of LITRV comprises 11,176 nucleo-tides (nt), contains non-coding 3′ and 5′ sequences, andfive main ORFs encoded in linear order (Figure 1A). Char-acteristic of rhabdoviruses, the coding regions are flankedby conserved sequences that likely serve as transcriptioninitiation and transcription termination/polyadenylationLtd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Figure 1 Organization of the LITRV genome. A) Schematic representation of LITRV ORFs. B) LITRV coding regions and corresponding transcriptionregulatory sequences.Tokarz et al. Virology Journal 2014, 11:26 Page 2 of 5http://www.virologyj.com/content/11/1/26signals. The polyadenylation signal consists of 3′- GAACUUUUUUU, which is followed by a 2 nt intergenicsequence and a putative transcription initiation sequence3′-UUGUU(U/G)N(G/A/U)U (Figure 1B). Homologysearch of all five coding regions revealed that each ORF ismost similar to the corresponding ORF of MOUV(Table 1). Both MOUV and LITRV cluster together andform a distinct phylogenetic clade within Rhabdoviridae(Figure 2). Similar to MOUV, only ORFs 1, 4 and 5, encod-ing the putative N, G and L proteins display homology tocorresponding Rhabdoviridae proteins. ORFs 2 and 3, en-coding the putative P and M proteins display no homologyto any rhabdovirus proteins outside of MOUV. The LITRVgenome also contains four alternative ORFs located withinthe P, M and G ORFs. These ORFs were designated P’, M’and G1’ and G2’, and would encode proteins of 81, 98, 106and 105 amino acids (aa), respectively, with no significantsequence identity with any other Rhabdoviridae protein byBLASTp analysis. Of the four ORFs, only P’ and G2’ havean initiation codon in suitable context for translation.As in all rhabdoviruses, the 3′ and 5′ terminal non-coding regions of LITRV contain partially complementaryregions, particularly at the terminal ends. The 3′ leader se-quence is 49 nt long while the length of the 5′ trailer se-quence is 115 nt. The terminal portions of the non-codingsequences are highly conserved in both LITRV andMOUV with 13 out of 19 nt identical in both viruses.Table 1 Comparison of LITRV and MOUV ORFsORFN PLITRV 1413 nt 831 nt470 aa (37.2%)* 276 aa (20.8%)MOUV 1404 nt 870 nt467 aa 289 aa*Indicates % amino acid identity of LITRV to MOUV.ORF analysisThe 1413 nt ORF1 is predicted to encode a nucleopro-tein of 470 aa that is 38% similar to MOUV N and con-tains many conserved rhabdovirus N domains includingthe RNA binding motif SPYS (Table 1). ORF2, compris-ing 831 nt, and, in accordance with its position withinthe rhabdoviral genome, is predicted to encode a 276 aaphosphoprotein. This protein shares 22.3% identity withORF2 of Moussa virus and is predicted to contain 17serine, 6 threonine and 1 tyrosine potential phosphoryl-ation sites (http://www.cbs.dtu.dk/services/NetPhos/). The618 nt ORF3 is predicted to encode a 206 aa protein. Theprotein shares 29.4% identity with MOUV ORF3. Noother known motifs or domains were recognized, althougha polyproline region, consisting of 8 consecutive prolineresidues was identified at aa 43–50. ORF 4, comprising1521 nt, is predicted to encode a 506 aa class I transmem-brane glycoprotein with 29.4% aa identity to MOUV Gprotein. The protein sequence contains a predicted N ter-minal 18 aa signal peptidase cleavage site, a hydrophobictrans-membrane domain at positions aa 458–480 followedby a C terminal 26 aa tail. It also contains 5 potential N-glycosylation sites at aa positions 58, 345, 359, 387, 424and contains all 12 cysteine residues conserved in otherrhabdoviruses. The 6396 nt L ORF encodes the RNA-dependent RNA polymerase, that in LITRV is predicted toencode a 2131 aa protein. The LITRV L is 51.5% identicalM G L618 nt 1521 6396205 aa (28.2%) 506 aa (28.5%) 2131 aa (51.7%)726 nt 1581 6428 nt241 aa 526 aa 2141 aaFigure 2 Phylogeny of LITRV. Maximum likelihood phylogenetic tree based on full length L protein sequences of currently recognized speciesof Rhabdoviridae. Gray boxes represent ICTV-accepted species within the genus. LITRV is indicated by *. Accession numbers are provided next tothe viral names. Due to excessive divergence, only the type species for Cytorhabdovirus, Novirhabdovirus and Nucleorhabdovirus are included. TheVesiculovirus piry virus is not included due to limited available sequence.Tokarz et al. Virology Journal 2014, 11:26 Page 3 of 5http://www.virologyj.com/content/11/1/26to MOUV and contains all the highly conserved residuesof negative strand RNA polymerases [4].To examine the presence of LITRV in ticks, wescreened archived cDNA generated from 50 individualadult A. americanum by polymerase chain reaction. Theticks were collected in April of 2008 in the same loca-tion as the ticks from the current study. LITRV sequencewas successfully amplified from one tick.DiscussionRhabdoviridae is a large family that includes over 100 vi-ruses classified together on the basis of genetic, serologicalor morphological analysis. The advent of high-throughputsequencing has resulted in genome characterization ofmany novel and archived rhabdoviruses revealing the vastdiversity of this family [3,5-10]. In this study we presentthe first complete genomic sequence of a tick-associatedrhabdovirus from the Western hemisphere. Although ar-thropods are frequently implicated as hosts of rhabdovi-ruses, thus far relatively few of these viruses have beenassociated with ticks [1,5,11]. We detected LITRV in A.americanum, a common tick species with a broad rangespanning eastern and south-central states in the US.Along with Ixodes scapularis and Dermacentor variablis,A. americanum is the primary human-biting hard tick inthe eastern part of the US and is implicated in transmis-sion of Ehrlichia species, Borrelia lonestari, Francisellatularensis, and the Heartland virus [12,13].Additional studies are needed to determine if A. amer-icanum is the primary host for LITRV or if other arthro-pods, and particularly, other ticks, have a role in itsenzoonotic transmission. While sequences were obtainedTokarz et al. Virology Journal 2014, 11:26 Page 4 of 5http://www.virologyj.com/content/11/1/26form A. americanum, this does not definitively implicateA. americanum as a biological host for LITRV. Detec-tion of microbial nucleic acids in organisms other thantheir presumed biological hosts has been reported andin a hematophagus organism these can represent nucleicacid remains of microbes acquired as part of a bloodmeal [14,15]. However, our detection of LITRV in A.americanum collected at the same site five years apart isconsistent with a role for this tick species in the lifecycle of this virus. Furthermore, deep sequencing ana-lysis of multiple pools of I. scapularis and D. variabiliscollected from the same location and did not identifyany LITRV-like sequences in these two tick species. Ourinitial survey of LITRV in individual A. americanum sug-gests that the prevalence of this virus may be low in tickpopulations within the examined area. Molecular surveysanalyzing different life stages of A. americanum and othertick species in broader geographical areas are necessary toestablish the range and prevalence of LITRV.Phylogenetically, LITRV forms a distinct clade withMOUV. MOUV was isolated from multiple mosquitopools in Cote d’Ivoire, suggesting that arthropods arethe likely hosts to this clade of rhabdoviruses. LITRVand MOU share many genetic similarities, includingidentical polyadenylation and terminal regions, as wellas similar conserved 3′ and 5′ termini sequences. Thehigh amino acid divergence of LITRV and MOUV rela-tive to other rhabdoviruses suggests that both viruses areunlikely to be associated with any of the current ascribedgenera and likely represent a unique taxonomic groupwithin Rhabdoviridae. We anticipate that continued sur-veillance of arthropod vectors may uncover other mem-bers of this clade.ConclusionsUsing high-throughput sequencing analysis of tick vir-omes, we discovered a novel tick associated rhabdovirus.This virus, which we provisionally name Long Island tickrhabdovirus, may represent a novel species within familyRhabdoviridae.Materials and methodsNucleic acid extractionAdult ticks were collected in Heckscher State Park (SuffolkCounty, NY) in April, 2013. Ticks were pooled and ho-mogenized in 500 μl of phosphate buffered saline. Five tickpools were analyzed; one pool of A. americanum (N = 25),and two pools each (N = 30/pool) of I. scapularis and D.variablis. The homogenate was purified through 0.22 μMfilter and treated with RNaseA and TurboDNase exonu-cleases. 250 μl of the filtrate was added to 750 μl of Nucli-Sens buffer and total nucleic acid (TNA) was extractedwith the EasyMag extraction platform (Biomerieux). TNAwas eluted in 35 μl volume followed by DNase treatment.Unbiased high throughput sequencingTotal nucleic acid was subjected to first and secondstrand cDNA synthesis with Super Script III reversetranscriptase (Invitrogen) and Klenow Fragment (NewEngland Biolabs), respectively. Ion Shear™ Plus ReagentsKit (Life Technologies) was used for double strandedcDNA fragmentation. Ion Xpress™ Adapters and uniqueIon Xpress™ Barcodes (Life Technologies) were ligated tofragmented material by using the Ion Plus FragmentLibrary kit, which also contained reagents for amplifica-tion of barcoded libraries. Ion OneTouch™ 200 TemplateKit v2 (Life Technologies) was used to bind barcoded li-braries to Ion Sphere™ particles (ISPS). Emulsion PCR ofDNA linked ISPS was performed on the Ion OneTouch™2 instrument (Life Technologies). Ion OneTouch™ ES in-strument was used to isolate template-positive ISPS. IonPGM™ Sequencing 200 Kit v2 (Life Technologies) wasused for sequencing of templated ISPS which wereloaded on the Ion 316™ Chip for further processing onthe Ion Personal Genome Machine® (PGM™) System(Life Technologies).The de-multiplexed reads were preprocessed by trim-ming primers and adaptors, length filtering, and maskingof low complexity regions (WU-BLAST 2.0). The re-maining reads were subjected to homology search usingBLASTn against a host genome database. The host-subtracted reads were assembled using the Newbler as-sembler (454, v2.6). Contigs and singletons were thensubjected to a homology search against the entire Gen-Bank database using BLASTn and the viral GenBankdatabase using BLASTx. Contigs and singletons withsimilarity to viral sequences from the BLASTx analysiswere again subjected to a homology search against entireGenBank database to correct for biased e-values. For po-tential viral candidates, close relatives were used to iden-tify low homology regions in the genome from BLASTx.Overall, out of approximately 190,000 sequence readsobtained by HTS, 96 reads with a mean length of 182 ntwere unique to LITRV. Gaps were filled in by PCR usingprimers specific to the assembled sequence. The finalsequence was verified by classical dideoxy sequencingusing primers designed to generate overlapping PCRproducts. Genomic termini were obtained by 5′ and 3′RACE kits (CloneTech). Genome assembly was per-formed with Geneious v 6.1. All phylogenetic trees wereconstructed with Mega 5.2 software.Tick screeningTo assess the presence of Long Island tick rhabdovirusin ticks, we used cDNA generated from adult A. ameri-canum ticks collected in April, 2008 at the same loca-tion. Tick cDNA was screened by PCR with primers5′-GGGACGATGCTCTAGTCACG-3′ (fwd), and 5′-TTTGTCTGTGAGGTCGGACG-3′ (rev) targeting aTokarz et al. Virology Journal 2014, 11:26 Page 5 of 5http://www.virologyj.com/content/11/1/26299 bp fragment of the N gene. PCR products wereassessed by gel electrophoresis and sequenced to confirmthat they represent Long Island tick rhabdovirus.The complete genome sequence of LITRV was depos-ited in Genebank under accession number KJ396935.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsRT and WIL conceived the study, analyzed data and wrote the manuscript.SS and MSL performed all assays. KJ performed all bioinformatics analysis. Allauthors read and approved the final manuscript.AcknowledgmentsThis work was supported by grants from the National Institutes of HealthAI057158 (Northeast Biodefense Center-Lipkin), USAID PREDICT and theDefense Threat Reduction Agency.Received: 15 January 2014 Accepted: 10 February 2014Published: 11 February 2014References1. Kuzmin IV, Novella IS, Dietzge RG, Padhi A, Rupprecht CE: Therhabdoviruses: biodiversity, phylogenetics, and evolution. Infect GenetEvol 2009, 9(4):541–553.2. King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ: Virus Taxonomy; NinthReport of the International Committee on Taxonomy of Viruses.Amsterdam, The Netherlands: Elsevier Academic Press; 2012.3. Quan PL, Junglen S, Tashmukhamedova A, Conlan S, Hutchinson SK, KurthA, Ellerbrok H, Egholm M, Briese T, Leendertz FH, Lipkin WI: Moussa virus: anew member of the Rhabdoviridae family isolated from Culex decensmosquitoes in Cote d’Ivoire. Virus Res 2010, 147(1):17–24.4. Poch O, Blumberg BM, Bougueleret L, Tordo N: Sequence comparison offive polymerases (L proteins) of unsegmented negative-strand RNAviruses: theoretical assignment of functional domains. J Gen Virol 1990,71(Pt 5):1153–1162.5. Ghedin E, Rogers MB, Widen SG, Guzman H, Travassos da Rosa AP, WoodTG, Fitch A, Popov V, Holmes EC, Walker PJ, Vasilakis N, Tesh RB: Kolentevirus, a rhabdovirus species isolated from ticks and bats in the Republicof Guinea. J Gen Virol 2013, 94(Pt 12):2609–2615.6. Kading RC, Gilbert AT, Mossel EC, Crabtree MB, Kuzmin IV, Niezgoda M,Agwanda B, Markotter W, Weil MR, Montgomery JM, Rupprecht CE,Miller BR: Isolation and molecular characterization of Fikirinirhabdovirus, a novel virus from a Kenyan bat. J Gen Virol 2013,94(Pt 11):2393–2398.7. Palacios G, Forrester NL, Savji N, Travassos da Rosa, Guzman H, Detoy K,Popov VL, Walker PJ, Lipkin WI, Vasilakis N, Tesh RB: Characterizationof Farmington virus, a novel virus from birds that is distantlyrelated to members of the family Rhabdoviridae. Virol J 2013,10:219.8. Vasilakis N, Widen S, Mayer SV, Seymour R, Wood TG, Popov V, Guzman H,Travassos da Rosa, Ghedin E, Holmes EC, Walker PJ, Tesh RB: Niakha virus: anovel member of the family Rhabdoviridae isolated from phlebotominesandflies in Senegal. Virology 2013, 444(1–2):80–89.9. Quan PL, Williams DT, Johansen CA, Jain K, Petrosov A, Diviney SM,Tashmukhamedova A, Hutchinson SK, Tesh RB, Mackenzie JS, Briese T, LipkinWI: Genetic characterization of K13965, a strain of Oak Vale virus fromWestern Australia. Virus Res 2011, 160(1–2):206–213.10. Stone DM, Kerr RC, Hughes M, Radford AD, Darby AC: Characterisation ofthe genomes of four putative vesiculoviruses: tench rhabdovirus, grasscarp rhabdovirus, perch rhabdovirus and eel rhabdovirus European X.Arch Virol 2013, 158(11):2371–2377.11. Labuda M, Nuttall PA: Tick-borne viruses. Parasitology 2004,129(Suppl):S221–S245.12. Goddard J, Varela-Stokes AS: Role of the lone star tick, Amblyommaamericanum (L.), in human and animal diseases. Vet Parasitol 2009,160(1–2):1–12.13. Savage HM, Godsey MS, Lambert A, Panella NA, Burkhalter KL, Harmon JR,Lash RR, Ashley DC, Nicholson WL: First detection of heartland virus(Bunyaviridae: Phlebovirus) from field collected arthropods. Am J TropMed Hyg 2013, 89(3):445–452.14. Telford SR, Wormser GP: Bartonella spp. transmission by ticks notestablished. Emerg Infect Dis 2010, 16(3):379–384.15. Whitehouse CA: Crimean-Congo hemorrhagic fever. Antiviral Res 2004,64(3):145–160.doi:10.1186/1743-422X-11-26Cite this article as: Tokarz et al.: Genome characterization of Long Islandtick rhabdovirus, a new virus identified in Amblyomma americanumticks. Virology Journal 2014 11:26.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submit

AC: Characterisation of the genomes of four putative vesiculoviruses: tench rhabdovirus, grass carp rhabdovirus, perch rhabdovirus and eel rhabdovirus European X. Arch Virol

BR: Isolation and molecular characterization of Fikirini rhabdovirus, a novel virus from a Kenyan bat. JG e nV i r o l2013, 94(Pt 11):2393–2398.

CE: The rhabdoviruses: biodiversity, phylogenetics, and evolution. Infect Genet Evol

Crimean-Congo hemorrhagic fever. Antiviral Res 2004, 64(3):145–160. doi:10.1186/1743-422X-11-26 Cite this article as: Tokarz et al.: Genome characterization of Long Island tick rhabdovirus, a new virus identified in Amblyomma americanum ticks.

EJ: Virus Taxonomy; Ninth Report of the International Committee on Taxonomy of Viruses.

RB: Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea.

RB: Niakha virus: a novel member of the family Rhabdoviridae isolated from phlebotomine sandflies in Senegal. Virology

Tesh RB: Characterization of Farmington virus, a novel virus from birds that is distantly related to members of the family Rhabdoviridae. Virol J

Tick-borne viruses. Parasitology

Tordo N: Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains.

Varela-Stokes AS: Role of the lone star tick, Amblyomma americanum (L.), in human and animal diseases. Vet Parasitol

WI: Genetic characterization of K13965, a strain of Oak Vale virus from Western Australia. Virus Res

WI: Moussa virus: a new member of the Rhabdoviridae family isolated from Culex decens mosquitoes in Cote d’Ivoire. Virus Res

WL: First detection of heartland virus (Bunyaviridae: Phlebovirus) from field collected arthropods. Am J Trop Med Hyg

Wormser GP: Bartonella spp. transmission by ticks not established. Emerg Infect Dis

https://academiccommons.columbia.edu/doi/10.7916/D8G451SC/download

Genome characterization of Long Island tick rhabdovirus, a new virus identified in Amblyomma americanum ticks

Abstract

Similar works

Full text

Available Versions

Crossref

Columbia University Academic Commons