Lwande et al. Parasites & Vectors 2014, 7:542http://www.parasitesandvectors.com/content/7/1/542

RESEARCH Open Access

Whole genome phylogenetic investigation of aWest Nile virus strain isolated from a tick sampledfrom livestock in north eastern KenyaOlivia Wesula Lwande1,2*, Marietjie Venter2,7, Joel Lutomiah3, George Michuki5, Cecilia Rumberia5, Francis Gakuya4,Vincent Obanda4, Caroline Tigoi1, Collins Odhiambo1, Fredrick Nindo6, Samwel Symekher3 and Rosemary Sang1,3

Abstract

Background: West Nile virus (WNV) has a wide geographical distribution and has been associated to causeneurological disease in humans and horses. Mosquitoes are the traditional vectors for WNV; however, the virus hasalso been isolated from tick species in North Africa and Europe which could be a means of introduction and spreadof the virus over long distances through migratory birds. Although WNV has been isolated in mosquitoes in Kenya,paucity of genetic and pathogenicity data exists. We previously reported the isolation of WNV from ticks collectedfrom livestock and wildlife in Ijara District of Kenya, a hotspot for arbovirus activity. Here we report the full genomesequence and phylogenetic investigation of their origin and relation to strains from other regions.

Methods: A total of 10,488 ticks were sampled from animal hosts, classified to species and processed in pools ofup to eight ticks per pool. Virus screening was performed by cell culture, RT-PCR and sequencing. Phylogeneticanalysis was carried out to determine the evolutionary relationships of our isolate.

Results: Among other viruses, WNV was isolated from a pool of Rhipicephalus pulchellus sampled from cattle,sequenced and submitted to GenBank (Accession number: KC243146). Comparative analysis with 27 different strainsrevealed that our isolate belongs to lineage 1 and clustered relatively closely to isolates from North Africa andEurope, Russia and the United States. Overall, Bayesian analysis based on nucleotide sequences showed thatlineage 1 strains including the Kenyan strain had diverged 200 years ago from lineage 2 strains of southern Africa.Ijara strain collected from a tick sampled on livestock was closest to another Kenyan strain and had diverged20 years ago from strains detected in Morocco and Europe and 30 years ago from strains identified in the USA.

Conclusion: To our knowledge, this is the first characterized WNV strain isolated from R. pulchellus. Theepidemiological role of this tick in WNV transmission and dissemination remains equivocal but presents tick versesmosquito virus transmission has been neglected. Genetic data of this strain suggest that lineage 1 strains fromAfrica could be dispersed through tick vectors by wild migratory birds to Europe and beyond.

Keywords: West Nile virus, Tick, Kenya, Livestock, Wildlife

BackgroundWest Nile virus (WNV) is classified in the familyFlaviviridae, genus Flavivirus which is closely relatedto viruses such as Japanese encephalitis, Saint Louis en-cephalitis, Usutu, Kunjin, Kokobera, Stratford, Alfuy andMurray Valley encephalitis that belong to the Japanese

* Correspondence: olwande@icipe.org1International Centre of Insect Physiology and Ecology, Nairobi, Kenya2Department of Medical Virology, University of Pretoria, Pretoria, South AfricaFull list of author information is available at the end of the article

© 2014 Lwande et al.; licensee BioMed CentraCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.

encephalitis serocomplex [1]. WNV has a wide geograph-ical distribution mainly in Africa, Europe, Russia, theMiddle East, India, Australia and North and South Americaand the Caribbean [2-4]. Recent outbreaks of WNV havebeen reported in Israel, France, Italy, Greece, South Africa,Hungary, southeast Romania and the USA [5-10].WNV is known to be transmitted primarily by mos-

quitoes of the genus Culex (principally C. univittatusand C. pipiens) [11-14] although evidence of tick-bornetransmission has also been documented [15,16]. Vector

l Ltd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), 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,

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competence studies performed mainly on soft ticks suchas Argas persicus, A. hermanni [17] and Ornithodorosmoubata [15] indicate the potential role of ticks in WNVtransmission. Although vector competence studies indi-cate a potential role for ticks in WNV transmission, re-sults of these studies indicate that ticks are likely to be farless efficient vectors of WNV than mosquitoes [15]. Theability of WNV to replicate in mosquitoes, mammals andbirds provides an opportunity for this virus to amplifyacross a wider geographical coverage enabling the circula-tion of different strains across continents.WNV causes mainly mild febrile illness but can result

in meningo-encephalitis, acute paralysis and death in se-vere cases in humans and horses [7,18-22]. In humans,the proportion of elderly patients presenting with severeneurologic illness due to WNV has been reported to behigh in the USA [23,24].WNV genome is composed of an 11 kb positive RNA

fragment that is translated to form a polyprotein. Thispolyprotein consists of three structural (C, prM/M and E)and seven non-structural proteins (NS1, NS2A, NS2B,NS3, NS4A, NS4B and NS5) [25]. Phylogenetically, WNVbelongs to two major lineages, Lineage 1 and 2. Lineage 1is composed of three sub-lineages a, b and c. Lineage1a is distributed in Africa, the Middle East, Europe andAmerica. Lineage 1b, which is also known as Kunjin virus(KUNV), is found in Australia and Linegae 1c mainlycirculates in India. WNV Lineage 2 comprises strainsisolated in sub-Saharan Africa [26]. Other proposed WNVlineages include Lineage 3, also known as Rabensbergvirus, that occurs in southern Moravia and Czech Repub-lic, Lineage 4 from Russia, Lineage 5 from India andLineage 6 that is also known as Koutango virus [27-29].Previous studies indicate that WNV circulates in Kenya,

having been isolated from diverse mosquito species [30-33].Evidence of natural isolation of WNV from Rh. pulchellusticks sampled from cattle and warthog has been docu-mented in Kenya [34], although there is scanty genetic andpathogenicity data on this virus since it has not been char-acterized in Kenya. In addition, the genetic profile andphylogeny of the circulating strains remains unresolved.Therefore the objectives of this study were (i) to deter-mine the role of ticks in the maintenance and circulationof arboviruses in Kenya and (ii) to genetically analyzethe isolated strains. This study was carried out during2010–2012 in Ijara district, a semi-arid pastoral regionwhich periodically experiences RVFV outbreaks that tendto co-circulate with other mosquito-borne arboviruseslike WNV.This study aimed to investigate circulation, transmis-

sion and diversity of WNV in Ijara District and test thehypothesis of its spread through migratory birds and tickvectors across the continent. This study will also aid inassessing the risk of disease, monitoring emerging tick-

borne viral diseases and providing a better understand-ing of the patterns and processes of evolution.

MethodsThis was a field-based descriptive cross-sectional andlaboratory-based study conducted between 2010 and 2012.

Ethics statementThis study was approved by the Kenya Medical ResearchInstitute Ethical Review Committee protocol number SSC2050. The Committee of the Department of Veterinaryand Capture Services (DVCS) of the Kenya Wildlife Service(KWS) approved the study including animal immobi-lization capturing protocols. KWS guidelines on WildlifeVeterinary Practice-2006 were followed. All KWS veteri-narians were guided by the Veterinary Surgeons Act Cap366 Laws of Kenya that regulates veterinary practices inKenya. For purposes of livestock use, permission was ob-tained from the owners involved. We worked in colla-boration with the department of veterinary services andveterinarians mandated by the government to do livestocksampling and research. The above terms were stipu-lated well in an agreement between the farmers andthe International Centre of Insect Physiology and Ecology(icipe), the hosting institution for the Arbovirus Incidenceand Diversity (AVID) Project Consortium.

Study areaThis study was carried out in Ijara District, North EasternProvince of Kenya. The district lies between latitude 1°7′Sand 2°3′S and longitude 40°4′E and 41°32′E. This is a lowaltitude (ranging between 0 and 90 meters above sea level)arid and semi-arid region where 90% of the people prac-tice nomadic pastoralism, keeping indigenous cattle, goats,sheep, donkeys and camels. Approximately one-quarter ofthe district is covered by the Boni forest bordering theIndian ocean, which is an indigenous open canopy forestthat forms part of the Northern Zanzibar-Inhamdare coastalforest mosaic. A section of the forest, the Boni NationalReserve is under the management of the Kenya WildlifeService as a protected conservation area and is hometo a range of wildlife species, including hirola antelope(also known as Hunter’s hartebeest), reticulated giraffe,elephant, buffalo, lion, leopard, cheetah, African wild dog,lesser kudu, desert warthog and bushbuck.

Sampling of ticksTicks sampling from both domestic animals and wildlifewas undertaken at various sites of the Ijara District,including the Boni National Game Reserve as describedpreviously in [34]. Qualified animal handlers who wore thenecessary protective gear (such as gloves, coveralls withtrouser cuffs taped to shoes, high-top shoes, socks pulledover trouser cuffs, and long-sleeved shirts) performed tick

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collections. Livestock (goats, sheep and cattle) were physic-ally restrained, whereas Kenya Wildlife Service veterinar-ians immobilized the wild animals (giraffe, warthog, lesserkudu and zebra) using a combination of etorphine hydro-chloride (M99R, Novartis, South Africa) and xylazinehydrochloride (Kyron, South Africa). Both livestock andwild animals were visually examined for ticks, with specialattention to the abdomen, back, anal area, and hind legs.If found, the ticks were pulled off manually, placed insterile plastic vials, and transported to the laboratoryin dry ice.

Tick identification and processingTick identification and processing was performed asdescribed previously in [34]. The ticks were washed twicewith sterile water to remove excess particulate conta-mination from animal skin, rinsed once with 70% ethanol,and then rinsed twice with minimum essential medium(MEM) containing antimicrobial agents (100U/mL peni-cillin, 100 lg/mL streptomycin and 1 μL/mL amphotericinB). Tick identification was performed using appropriateidentification keys. The ticks were transferred to sterilevials and stored at -80°C until processed for virus isola-tion. Ticks were later thawed in ice (4°C), identified andpooled into groups of one to eight (depending on size) byspecies, sex, and animal host. Each pool was homogenizedusing 90-mesh alundum in a prechilled, sterile mortar andpestle with 1.6–2 mL ice-cold MEM containing 15% fetalbovine serum (FBS), 2% glutamine, 100U/mL penicillin,100 lg/mL streptomycin, and 1 μL/mL amphotericin B.The homogenates were clarified by low-speed centrifuga-tion at 1500 rpm for 15 min at 4°C, and supernatants ali-quoted and stored at -80°C. In the case of Hyalommaspp., the primary vectors of Crimean–Congo hemorrhagicfever virus (CCHFV), each pool was prescreened forCCHF by reverse transcription-polymerase chain reac-tion (RT-PCR) to exclude this virus prior to cell cul-ture screening.

Virus isolationVirus isolation was performed as described in [34]. Briefly,Vero cells were grown in 25-cm2 cell culture flasks to 80%confluency in MEM containing 10% FBS, 2% glutamine,100U/mL penicillin, 100 lg/mL streptomycin, and 1 μ/mLamphotericin B. The cells were then rinsed with sterilephosphate-buffered saline (PBS) and 0.2 mL of clarifiedtick homogenate was added followed by incubation at37°C for 45 min to allow virus adsorption. After incuba-tion, MEM supplemented with 2% FBS, 2% glutamine,100 U/mL penicillin, 100 lg/mL streptomycin, and 1 μ/mLamphotericin B was added into the flasks and the cellsallowed to incubate at 37°C for 14 days while observingcytopathic effect on a daily basis. The supernatants ofvirus-infected Vero cell cultures exhibiting cytopathic

effect of approximately 70% were harvested from theflasks for virus identification. The pooled infection rateprogram (PooledInfRat, Centers for Disease Controland Prevention, Fort Collins, CO; http://www.cdc.gov/ncidod/dvbid/westnile/software.htm/) was used to com-pare virus infection rates in the tick species collectedand processed in this study.

RNA sample preparation for 454 sequencingTick homogenates with positive cytopathic effect were fil-tered through a 0.22 μm filter, followed by RNA extractionusing TRIzol reagent (Invitrogen). RNA was amplifiedusing the modified random priming mediated sequenceindependent single primer amplification (RP-SISPA) me-thodology [35].

454 sequencingEach amplified sample was further processed as describedfor shotgun library preparation in GS FLX 454 technology.The sequencing reads were trimmed to remove SISPAprimers and barcodes and only reads with a length greaterthan 50 bp were retained. Low complexity repeatswere masked using Repeatmasker (Repeat-Masker Open-3.0.1996-2010 http://www.repeatmasker.org) and sequenceswith more than 50% repeats were excluded. The sequencesin each pool were assembled using the Newbler assemblerversion 2.5.3 with default settings (Roche. Genome Se-quencer FLX Data Analysis Software Manual. Mannheim,Germany: Roche Applied Science, 2007). Contiguous se-quences and reads which did not assemble into contigswere categorized using BLASTN and BLASTX homologysearches against the non-redundant nucleotide and aminoacid databases from NCBI (version June 2011). Taxonomicclassification of each contig/read was investigated usingMEGAN 4.0.

Phylogenetic analysisComparative phylogenetic analyses were performed todescribe and infer the lineage and occurrence of anyselective pressures (amino acid substitutions) in the WNVstrain isolated from R. pulchellus ticks in this survey inrelation to strains isolated from other vectors (available inGenBank). The assembled sequence data isolated fromticks collected in Ijara District, Kenya were combined withavailable global full genome WNV reference sequences re-trieved from the NCBI GenBank database. The final data-set comprised of 28 full genome sequences approximately11,000 bp long. These were aligned using MUSCLE mul-tiple sequence alignment program and manually editedand visualized using MacClade v4.08a [36]. The generaltime reversible (GTR) nucleotide substitution model withfour gamma rate heterogeneity categories was foundto be appropriate for these data based on the Modeltestanalysis [37].

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Phylogenies were obtained using RAxML v7.4.4 [38]implementing the GTR model of nucleotide substitu-tions with four gamma categories of rate heterogeneity,a method for phylogenetic tree inference using maximumlikelihood/rapid bootstrapping techniques for long se-quences. Statistical support for the clustering observedwas assessed using bootstrapping techniques engraved inthe RAxML algorithm.To infer the evolutionary relationships among the

selected WNV strains isolated from different vectors,dated ancestral states were reconstructed using BEASTavailable at (http://beast.bio.ed.ac.uk) under the GTR +gamma and proportion of invariant sites. BEAST em-ploys Bayesian Markov Chain Monte Carlo MCMC treeestimation strategies. Two independent chain runs wereconducted for 100 million generations, sampling treesand parameters at intervals of 10,000 generations underthe uncorrelated lognormal relaxed clock model. Togauge the convergence of the runs, the log files werevisualized in Tracer software included in the BEASTsoftware package.Clade support was assessed by calculating posterior

probability for monophyletic clades observed in the treetopology. The trees obtained were summarised usingTreeannotaor program included in BEAST software pack-age with the first 10% trees obtained before the conver-gence of the runs discarded as “burn-in”.

Amino acid substitution (selection) analysisComprehensive site-specific amino acid substitution ana-lysis was done using SLAC and FUBAR methods availablein Hyphy suite of selection pressure analysis tools ac-cessible through data-monkey web server as (http://www.hyphy.org/w/index.php/SLAC and http://www.hyphy.org/w/index.php/FUBAR) for prediction of site specific aminoacid changes and genome distribution of sites under posi-tive and negative selection pressure, respectively [39,40].The alignment was first analyzed for presence of anyrecombination using GARD program also embedded inthe Hyphy and accessed through data-monkey web servergraphical user interface [41]. Selection analyses were per-formed under the GTR model of nucleotide substitutionand the significance of the prediction of the amino acidsat specific sites were assessed statistically by p-values <0.1and posterior probabilities ≥0.9 for SLAC and FUBARanalyses respectively [42].

ResultsA total of 10,488 ticks were sampled from both livestockand wild animal hosts and processed into 1,520 pools ofup to eight ticks per pool. WNV screening in tickscollected from various animal hosts in Ijara District,Kenya, resulted in two tick-borne isolates obtained from R.pulchellus sampled from cattle and warthog as described in

[34]. These isolates were confirmed to be WNV after beingsubjected to PCR testing and subsequent sequencing. TheseWNV isolates were subjected to Sanger sequencing of thenon-structural protein 5 (NS5) gene region (positions 9254to 9609) used for identification and confirmed to be WNV.One of the two isolates (ATH002316 GenBank accessionnumber; KC243146.1) was subjected to 454 sequencingusing random/shotgun sequencing to attempt full genomesequencing. Ninety-nine percent (99%) of the WNVgenome was obtained by 454 sequencing. WNV genomepositions 4 to 10,867 corresponding to polyprotein whichconsist of both structural (C, E and M) and non-structural(1, 2A, 2B, 3, 4A, 4B and 5) proteins and amino acid posi-tions 1–3,434 was obtained.

Phylogenetic clustering of the Kenyan Tick-borne WNVTwenty-six selected representative full or near full gen-ome WNV sequences obtained from different vectorsand corresponding clinical isolates from humans andanimals from other parts of Africa, the Americas, Asia,Australia, and Europe were combined with the Kenyanisolate to make a final alignment of 27 sequences andused for phylogenetic analyses. Maximum likelihoodphylogeny was obtained using RAxML program underGTR model of nucleotide substitution and bootstrap-ping with 1,000 replicates (Figure 1). The Kenyan strain(KC243146.1) clustered together with other strains fromdifferent parts of the world belonging to WNV Lineage 1.

Evolutionary relationships among WNV strainsThe evolutionary relationship of the Kenyan tick-borneWNV strain and other WNV strains was assessed usingBEAST tool under general time reversible (GTR) model.The clustering observed under this method was similar tothat observed under maximum likelihood providing furthersupport that the WNV isolate (KC243146.1) from ticks col-lected in Ijara district in Northern Kenya clustered closelyto Lineage 1 strains from Kenya as well as Europe, NorthAfrica and the USA. The time to the most recent commonancestor of Lineage 1 viruses was estimated to be approxi-mately 200 years (Figure 2) while the Ijara tick strain di-verged approximately 21 years ago from the other Kenyanstrain or European strains and 30 years ago from the USAstrains. The strains from North Africa were closest to thestrains studied in France and only diverged 9 years ago,suggesting this may have been the possible route of intro-duction to Europe. The Kenyan strain (KC243146.1) wasdistinct from Lineage 2 strains from southern Africa anddiverged more than 200 years ago from these.

Selection signatures (amino acid substitutions) inWNV genomeThe 27 WNV full genome sequences were first testedfor recombination. Two partitions of 1–980 codons and

Lineage 4

Lineage 3

Lineage 2

Lineage 1

Japanese encephalitis virus (out-group)

Kenyan isolate

Figure 1 (See legend on next page.)

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(See figure on previous page.)Figure 1 Maximum likelihood phylogeny of selected WNV sequences. GenBank accession numbers. Strain abbreviations (isolation source,country, year and accession number): SE-90: Mimomyia lacustris, Senegal, 1990, DQ318019; ATH002316, R. pulchellus, Kenya, 2010, KC243146.1;Ug-37: human, Uganda, 1937, AY532665; WNFCG: derivate of Ug-37, M12294; SA-89: human, South Africa, 1989, EF429197; SA-01: human, SouthAfrica, 2001, EF429198; Hu-04: Accipiter gentilis, Hungary, 2004, DQ116961; Gr-10: Culex pipiens, Greece, 2010, HQ537483; SA-58: human, SouthAfrica, 1958, EF429200; SA-00: human, South Africa, 2000, EF429199; Rus-07: human, Russia, 2007, FJ425721; Rab-97: Cx. pipiens, Czech Republic,1997, AY765264.1; CAR-72: Cx. tigripes, Central African Republic, 1972, DQ318020.1; Sarafend: derivate of Ug-37, AY688948.1; Coracopsis vasa,Madagascar, 1978, DQ176636.2; Rus-98: Dermacentor marginatus, Russia, 1998, AY277252.1; human brain in 1999, LEIV-Vlg99-27889, Russia: Volgograd,low Volga, AY277252.1; NY-99, USA, KC407666.1; total brain RNA (patient NYC99002), HNY1999, USA: New York, AF202541.1; crow, WN NY 2000-crow3356,USA: New York, AF404756.1; NY99,385-99, USA, DQ211652.1; Homo sapiens, Italy/2012/Livenza/31.1, Italy: Veneto region, Venice province, JX556213.1; brainof horse with encephalitis, 2003, Morocco, AY701413.1; brain, house sparrow (Passer domesticus), France 405/04, France, 2004, DQ786572.1; equine, WNItaly 1998-equine, Italy, AF404757.1; Culex univittatus, KN3829, AY262283.1 and swine brain, China, 2007, FJ495189.1. Japanese encephalitis virus isolateGenBank accession number FJ495189.1 was used an out-group.

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981–3650 codon sites were obtained; however, no re-combination signal was detected in any of the genomesused for these analyses. The Hyphy SLAC selection ana-lysis method predicted 21 codon sites to be under positiveselection based on significant p-values. (Figure 3). Site-specific amino acid changes as predicted from SLAC ana-lysis in Hphy datamonkey webserver were extracted andrecorded as shown in Additional file 1: Table S1.To better visualize the distribution of sites under selective

pressure, the distribution of predicted posterior probabil-ities for the respective codon sites along the WNV genomewere plotted and it was observed that there was differentialdistribution of positive and negative pressures along thevarious genomic regions of WNV strains (Figure 3).

DiscussionStudies conducted in Kenya during the RVF outbreaksdemonstrated the presence of WNV in diverse mosquitospecies sampled from Rift Valley and North-EasternProvinces. Although WNV has not been associated withany outbreak in Kenya, this virus has been isolated frommale C. univittatus sampled from Rift Valley [33], Aedessudanensis from North Eastern Kenya [32] and Culexspp. and Aedes spp. sampled from North Eastern Kenya[30]. Neutralizing antibodies against WNV have also beendetected in sera of diverse mammalian species includingcattle in Turkey, thus providing evidence of WNV circ-ulation in the region [43]. The isolation of WNV from R.pulchellus sampled from cattle in Ijara District of NorthEastern Province of Kenya suggests that this virus mightbe circulating amongst diverse vectors, animals andhumans in this region. Rhipicephalus pulchellus is themost predominant tick species in arid and semi-arid re-gions [44] and the most abundant in cattle of Boranapastoral areas [45]. This tick species has a three-host lifecycle and has been shown to infest domestic and largewildlife hosts such as cattle and zebra [44] and thereforethere is a possibility that it may serve as a potential reser-voir and vector of WNV and other arboviruses.Arbovirus surveillance studies conducted in Kenya have

shown that WNV is transmitted mainly by mosquitoes

belonging to the genus Culex [30,33]. Therefore, the dis-covery of WNV in R. pulchellus sampled from cattle inIjara District suggest its potential as a vector for longerdistance transfer. This study constitutes the first WNV de-tection in R. pulchellus ticks in Kenya. Vector competencestudies performed on Hyalomma marginatum in Portugalhighlight the role of ticks in the natural transmission ofWNV [46]. Ornithodoros moubata have also been foundto serve as potential reservoirs and vectors of WNV [15].Phylogenetic analysis of the 27 whole genome sequences

including tick-borne WNV isolate obtained from Ijaradistrict in Kenya showed that the Kenyan tick-borneWNV strain belongs to Lineage 1. Other African sequences,mostly those isolated from southern Africa, segregated toform a large cluster consisting of WNV Lineage 2 viruses.Despite being isolated from ticks, WNV strains isolatedfrom mosquitoes and ticks in Kenya formed distinct cladein the time scaled phylogeny (BEAST MCC tree) pointingto cross-species transmission of this virus in Kenya.Further phylogenetic evolutionary analysis shows that

the WNV strain (KC243146.1) isolated from Kenya is evo-lutionarily related as it clusters together in Bayesian phylo-genetic reconstruction. Further observations made fromthis analysis point to the Japanese encephalitis virus isolateas the most common recent ancestor of the WNV Lineage1 viruses with the divergence dating back approximately200 years. Full genome analysis also suggested thatthe Lineage 1 strains used in this study from Kenya,North Africa and Europe diverged approximately 20 yearsago which does not suggest that the Kenyan strain(KC243146.1) was involved in recent introduction. Theclosest ancestor to the European strains was from Moroccowhich diverged nine years ago from strains of France andItaly. This does emphasize a possible role of inter- andcross-continental spread for WNV strains. Transfer of ticksthrough migratory birds remains a potential mechanism toachieve this. Lineage 2 strains diverged more than 200 yearsago from Lineage 1. The South African strains diverged23 years ago from those in Europe. However the Greekstrain diverged only nine years ago from those in Hungary;this may suggest that the Central European strains were the

Lineage 3

Lineage 2

Lineage 1

Lineage 4

Japanese encephalitis virus (out-group)

Kenyan Isolate

Figure 2 Evolutionary relationships among the WNV strains. Maximum Clade Credibility tree generated under GTR gamma model ofnucleotide substitution with 4 gamma categories of rate heterogeneity and constant size coalescent population model assuming log-normalpriors. GenBank accession numbers: DQ318019, KC243146.1, AY532665, M12294, EF429197, EF429198, DQ116961, HQ537483, EF429200, EF429199,FJ425721, AY765264.1, DQ318020.1, AY688948.1, DQ176636.2, AY277252.1, AY277252.1, KC407666.1, AF202541.1, AF404756.1, DQ211652.1,JX556213.1, AY701413.1, DQ786572.1, AF404757.1, AY262283.1 and FJ495189.1. Node labels are median heights (in years).

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origin of this outbreak rather than recent introductionfrom Africa.WNV Lineage 1 strains have a widespread distribution

in Africa, Europe, the Middle East, America, Australiaand India. It is possible that WNV Lineage 1 could havebeen exported from Africa to Europe in particular, bymigratory birds that might have had ticks attached tothem though evidence of R. pulchellus infesting birds islacking. The phylogenetic analysis suggests that Moroccorather than Kenya was the source of recent introductionto Europe. Both highly and less neuroinvasive strains havebeen shown to exist in both Lineage 1 and 2 [7]. Experi-mental or clinical data on the neurovirulence of Lineage 1strains from Kenya are not yet available and investigationof WNV as a cause of neurological disease in humans andanimals in Kenya is warranted.Neurovirulence is associated with specific genotypes

and not related to lineage, source of isolate, geographicdistribution, passage level, or year of isolation [47-49]. Se-lection analyses carried out in the current study revealeddifferential selection pressures operating at different generegions along the WNV genome. In this analysis it wasobserved that non-synonymous amino acid substitutionsare more prevalent in 5′ structural genes region and lessprevalent in non-structural gene regions towards the 3′prime end. Of special interest is the observation that lim-ited or no diversifying selection pressures seemed to beoperating on the polymerase gene. This may be attributedto its strategic function in the replication of the virus gen-ome hence being kept under negative selection pressure

P

Posterior probability of amino acid substitution

Figure 3 Predicted codon site specific negative and positive selectionpositive selection are indicated in red and sites predicted to be under neg

as seen in Figure 3 (highlighted in blue). The amino acidchanges show the same trend in the viruses belonging tosimilar lineages further underscoring the importance ofthese genes in the maintenance of progeny, i.e. conferringviral fitness to the various strains. The Kenyan WNVisolate (KC243146.1) from ticks shows amino acid substi-tutions commonly observed in other strains belongingto Lineage 1 isolated from ticks and mosquitoes fromdifferent locations worldwide. These results resonate wellwith previous studies that have attempted to elucidategenetic markers for the virulence and pathogenicity of dif-ferent lineages of the WNV strains. According to a studyconducted by Beasley [50], enhanced virulence of NorthAmerican WNV strains compared with other Lineage 1strains of the Old World was linked to the envelope pro-tein glycosylation. WNV Lineage 2 strains isolated frompatients in South Africa all had these envelope protein gly-cosylation sites which have been associated with increasedvirulence [51].The isolation of WNV from R. pulchellus species sam-

pled from cattle in Ijara District might imply that eitherthis tick species may have acquired the virus after ingest-ing a viraemic blood meal from the infected animal orthe virus was present in the tick which may imply itspotential as vector, reservoir and disseminator of WNV.In general, birds are known as reservoirs of WNV whilemost mammals have relatively low viremia and act asdead-end hosts [3]. The role of cattle and warthogs asreservoirs for WNV is not known. Although thought tobe unlikely, this could be the source of infection to the

Codon position along the genome

ositive Negative

eee

(amino acid substitutions) along the WNV genome. Sites underative selection are indicated in blue.

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tick. Surveillance for arboviruses such as WNV shouldbe conducted and appropriate prevention and controlstrategies be put in place so as to be able to manage arbo-viruses early enough before they cause outbreaks [52].

ConclusionsThe identification of WNV Lineage 1 in Ijara Districtfurther elucidates the geographic range of Lineage 1 and2 strains in Africa. Lineage 2 strains have been describedin Tanzania and are almost exclusively found in SouthAfrica [2] while Lineage 1 strains are the major lineagereported in North Africa, Eurasia, India, the Middle Eastand the Americas. The close proximity of Europe toAfrica may be the reason that both lineages have emergedin Europe although the exact origin of importation ofLineage 2 strains to Europe is not known. Tick vectorspose a feasible transmission/dissemination mechanism forpreserving WNV over long distances and extended time ifconsidering the relatively short viremic period in mostanimals. The Kenyan strain identified here was not closeto other published WNV strains if considering the diver-gence time from known strains. This suggests that afurther uncharacterized source of WNV strains exist inlivestock and ticks in Africa.

Additional file

Additional file 1: Table S1. Amino acid substitutions of the WNVpolyprotein gene that corresponds to the structural (C, E and M) andnon-structural (1, 2A, 2B, 3, 4A, 4B and 5) proteins.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsOWL RS conceived and designed experiments. OWL conducted theexperimental work. OWL GM CR FN SS analyzed the data. OWL MV JL GM CRFG VO CT CO RS contributed to the manuscript. All authors approved thefinal version for submission.

AcknowledgementsWe wish to thank the Arbovirus Incidence and Diversity Project consortium-ledby International Centre of Insect Physiology and Ecology (ICIPE) and including;International Livestock Research Institute, Kenya Agricultural Research Institute,Ministry of Livestock Development-Central Veterinary Laboratories, KenyaMedical Research Institute, Ministry of Public Health and Sanitation and KenyaWildlife Service; The director of ICIPE, Prof. Christian Borgemeister, for providingall the necessary support in spearheading the project.

Author details1International Centre of Insect Physiology and Ecology, Nairobi, Kenya.2Department of Medical Virology, University of Pretoria, Pretoria, South Africa.3Kenya Medical Research Institute, Nairobi, Kenya. 4Kenya Wildlife Service,Nairobi, Kenya. 5International Livestock Research Institute, Nairobi, Kenya.6Computational Biology Group, Institute of Infectious Diseases and MolecularMedicine, (IIDMM) University of Cape Town, Cape Town, South Africa.7Global Disease Detection, United States-Centers for Disease Control, Pretoria,South Africa.

Received: 27 August 2014 Accepted: 15 November 2014

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Table 1: Amino acid substitutions of the WNV polyprotein gene that corresponds to the structural (C, E and M) and non-structural (1, 2A, 2B, 3, 4A, 4B and 5) proteins

NY99_USA_2005 M S K K P G G P G K S R A V N M L K R G M P R V L S L I G L [ 30]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 30]

NY99_USA_2005 K R A M L S L I D G K G P I R F V L A L L A F F R F T A I A [ 60]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 60]

NY99_USA_2005 P T R A V L D R W R G V N K Q T A M K H L L S F K K E L G T [ 90]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 90]

NY99_USA_2005 L T S A I N R R S S K Q K K R G G K T G I A V M I G L I A S [ 120]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . [ 120]

NY99_USA_2005 V G A V T L S N F Q G K V M M T V N A T D V T D V I T I P T [ 150]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 150]

NY99_USA_2005 A A G K N L C I V R A M D V G Y M C D D T I T Y E C P V L S [ 180]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 180]

NY99_USA_2005 A G N D P E D I D C W C T K S A V Y V R Y G R C T K T R H S [ 210]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 210]

NY99_USA_2005 R R S R R S L T V Q T H G E S T L A N K K G A W M D S T K A [ 240]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 240]

NY99_USA_2005 T R Y L V K T E S W I L R N P G Y A L V A A V I G W M L G S [ 270]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 270]

NY99_USA_2005 N T M Q R V V F V V L L L L V A P A Y S F N C L G M S N R D [ 300]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 300]

NY99_USA_2005 F L E G V S G A T W V D L V L E G D S C V T I M S K D K P T [ 330]

Additional file 1: Table S1..

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 330]

NY99_USA_2005 I D V K M M N M E A A N L A E V R S Y C Y L A T V S D L S T [ 360]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 360]

NY99_USA_2005 K A A C P T M G E A H N D K R A D P A F V C R Q G V V D R G [ 390]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 390]

NY99_USA_2005 W G N G C G L F G K G S I D T C A K F A C S T K A I G R T I [ 420]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [ 420]

NY99_USA_2005 L K E N I K Y E V A I F V H G P T T V E S H G N Y S T Q V G [ 450]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . [ 450]

NY99_USA_2005 A T Q A G R L S I T P A A P S Y T L K L G E Y G E V T V D C [ 480]

ATH002316_Kenya_2012 . . . . . . F . . . . . . . . . . . . . . . . . . . . . . . [ 480]

NY99_USA_2005 E P R S G I D T N A Y Y V M T V G T K T F L V H R E W F M D [ 510]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 510]

NY99_USA_2005 L N L P W S S A G S T V W R N R E T L M E F E E P H A T K Q [ 540]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 540]

NY99_USA_2005 S V I A L G S Q E G A L H Q A L A G A I P V E F S S N T V K [ 570]

ATH002316_Kenya_2012 . . . . . . . . G . R S C I K . W L E P S L W I . . . . . . [ 570]

NY99_USA_2005 L T S G H L K C R V K M E K L Q L K G T T Y G V C S K A F K [ 600]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 600]

NY99_USA_2005 F L G T P A D T G H G T V V L E L Q Y T G T D G P C K V P I [ 630]

ATH002316_Kenya_2012 . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . [ 630]

NY99_USA_2005 S S V A S L N D L T P V G R L V T V N P F V S V A T A N A K [ 660]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . [ 660]

NY99_USA_2005 V L I E L E P P F G D S Y I V V G R G E Q Q I N H H W H K S [ 690]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 690]

NY99_USA_2005 G S S I G K A F T T T L K G A Q R L A A L G D T A W D F G S [ 720]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 720]

NY99_USA_2005 V G G V F T S V G K A V H Q V F G G A F R S L F G G M S W I [ 750]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 750]

NY99_USA_2005 T Q G L L G A L L L W M G I N A R D R S I A L T F L A V G G [ 780]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 780]

NY99_USA_2005 V L L F L S V N V H A D T G C A I D I S R Q E L R C G S G V [ 810]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 810]

NY99_USA_2005 F I H N D V E A W M D R Y K Y Y P E T P Q G L A K I I Q K A [ 840]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 840]

NY99_USA_2005 H K E G V C G L R S V S R L E H Q M W E A V K D E L N T L L [ 870]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . [ 870]

NY99_USA_2005 K E N G V D L S V V V E K Q E G M Y K S A P K R L T A T T E [ 900]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 900]

NY99_USA_2005 K L E I G W K A W G K S I L F A P E L A N N T F V V D G P E [ 930]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 930]

NY99_USA_2005 T K E C P T Q N R A W N S L E V E D F G F G L T S T R M F L [ 960]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 960]

NY99_USA_2005 K V R E S N T T E C D S K I I G T A V K N N L A I H S D L S [ 990]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 990]

NY99_USA_2005 Y W I E S R L N D T W K L E R A V L G E V K S C T W P E T H [1020]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1020]

NY99_USA_2005 T L W G D G I L E S D L I I P V T L A G P R S N H N R R P G [1050]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1050]

NY99_USA_2005 Y K T Q N Q G P W D E G R V E I D F D Y C P G T T V T L S E [1080]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1080]

NY99_USA_2005 S C G H R G P A T R T T T E S G K L I T D W C C R S C T L P [1110]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . [1110]

NY99_USA_2005 P L R Y Q T D S G C W Y G M E I R P Q R H D E K T L V Q S Q [1140]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1140]

NY99_USA_2005 V N A Y N A D M I D P F Q L G L L V V F L A T Q E V L R K R [1170]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1170]

NY99_USA_2005 W T A K I S M P A I L I A L L V L V F G G I T Y T D V L R Y [1200]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1200]

NY99_USA_2005 V I L V G A A F A E S N S G G D V V H L A L M A T F K I Q P [1230]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1230]

NY99_USA_2005 V F M V A S F L K A R W T N Q E N I L L M L A A V F F Q M A [1260]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . M . . . . . . . . [1260]

NY99_USA_2005 Y H D A R Q I L L W E I P D V L N S L A V A W M I L R A I T [1290]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1290]

NY99_USA_2005 F T T T S N V V V P L L A L L T P G L R C L N L D V Y R I L [1320]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1320]

NY99_USA_2005 L L M V G I G S L I R E K R S A A A K K K G A S L L C L A L [1350]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1350]

NY99_USA_2005 A S T G L F N P M I L A A G L I A C D P N R K R G W P A T E [1380]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1380]

NY99_USA_2005 V M T A V G L M F A I V G G L A E L D I D S M A I P M T I A [1410]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1410]

NY99_USA_2005 G L M F A A F V I S G K S T D M W I E R T A D I S W E S D A [1440]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1440]

NY99_USA_2005 E I T G S S E R V D V R L D D D G N F Q L M N D P G A P W K [1470]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1470]

NY99_USA_2005 I W M L R M V C L A I S A Y T P W A I L P S V V G F W I T L [1500]

ATH002316_Kenya_2012 . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . [1500]

NY99_USA_2005 Q Y T K R G G V L W D T P S P K E Y K K G D T T T G V Y R I [1530]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1530]

NY99_USA_2005 M T R G L L G S Y Q A G A G V M V E G V F H T L W H T T K G [1560]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1560]

NY99_USA_2005 A A L M S G E G R L D P Y W G S V K E D R L C Y G G P W K L [1590]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1590]

NY99_USA_2005 Q H K W N G Q D E V Q M I V V E P G K N V K N V Q T K P G V [1620]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1620]

NY99_USA_2005 F K T P E G E I G A V T L D F P T G T S G S P I V D K N G D [1650]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1650]

NY99_USA_2005 V I G L Y G N G V I M P N G S Y I S A I V Q G E R M D E P I [1680]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1680]

NY99_USA_2005 P A G F E P E M L R K K Q I T V L D L H P G A G K T R R I L [1710]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1710]

NY99_USA_2005 P Q I I K E A I N R R L R T A V L A P T R V V A A E M A E A [1740]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1740]

NY99_USA_2005 L R G L P I R Y Q T S A V P R E H N G N E I V D V M C H A T [1770]

ATH002316_Kenya_2012 . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . [1770]

NY99_USA_2005 L T H R L M S P H R V P N Y N L F V M D E A H F T D P A S I [1800]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1800]

NY99_USA_2005 A A R G Y I S T K V E L G E A A A I F M T A T P P G T S D P [1830]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1830]

NY99_USA_2005 F P E S N S P I S D L Q T E I P D R A W N S G Y E W I T E Y [1860]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1860]

NY99_USA_2005 T G K T V W F V P S V K M G N E I A L C L Q R A G K K V V Q [1890]

ATH002316_Kenya_2012 I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1890]

NY99_USA_2005 L N R K S Y E T E Y P K C K N D D W D F V I T T D I S E M G [1920]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1920]

NY99_USA_2005 A N F K A S R V I D S R K S V K P T I I T E G E G R V I L G [1950]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1950]

NY99_USA_2005 E P S A V T A A S A A Q R R G R I G R N P S Q V G D E Y C Y [1980]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [1980]

NY99_USA_2005 G G H T N E D D S N F A H W T E A R I M L D N I N M P N G L [2010]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2010]

NY99_USA_2005 I A Q F Y Q P E R E K V Y T M D G E Y R L R G E E R K N F L [2040]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2040]

NY99_USA_2005 E L L R T A D L P V W L A Y K V A A A G V S Y H D R R W C F [2070]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2070]

NY99_USA_2005 D G P R T N T I L E D N N E V E V I T K L G E R K I L R P R [2100]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2100]

NY99_USA_2005 W I D A R V Y S D H Q A L K A F K D F A S G K R S Q I G L I [2130]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2130]

NY99_USA_2005 E V L G K M P E H F M G K T W E A L D T M Y V V A T A E K G [2160]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2160]

NY99_USA_2005 G R A H R M A L E E L P D A L Q T I A L I A L L S V M T M G [2190]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2190]

NY99_USA_2005 V F F L L M Q R K G I G K I G L G G A V L G V A T F F C W M [2220]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . [2220]

NY99_USA_2005 A E V P G T K I A G M L L L S L L L M I V L I P E P E K Q R [2250]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2250]

NY99_USA_2005 S Q T D N Q L A V F L I C V M T L V S A V A A N E M G W L D [2280]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2280]

NY99_USA_2005 K T K S D I S S L F G Q R I E V K E N F S M G E F L L D L R [2310]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2310]

NY99_USA_2005 P A T A W S L Y A V T T A V L T P L L K H L I T S D Y I N T [2340]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2340]

NY99_USA_2005 S L T S I N V Q A S A L F T L A R G F P F V D V G V S A L L [2370]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2370]

NY99_USA_2005 L A A G C W G Q V T L T V T V T A A T L L F C H Y A Y M V P [2400]

ATH002316_Kenya_2012 . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . [2400]

NY99_USA_2005 G W Q A E A M R S A Q R R T A A G I M K N A V V D G I V A T [2430]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2430]

NY99_USA_2005 D V P E L E R T T P I M Q K K V G Q I M L I L V S L A A V V [2460]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2460]

NY99_USA_2005 V N P S V K T V R E A G I L I T A A A V T L W E N G A S S V [2490]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2490]

NY99_USA_2005 W N A T T A I G L C H I M R G G W L S C L S I T W T L I K N [2520]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2520]

NY99_USA_2005 M E K P G L K R G G A K G R T L G E V W K E R L N Q M T K E [2550]

ATH002316_Kenya_2012 . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2550]

NY99_USA_2005 E F T R Y R K E A I I E V D R S A A K H A R K E G N V T G G [2580]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2580]

NY99_USA_2005 H P V S R G T A K L R W L V E R R F L E P V G K V I D L G C [2610]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2610]

NY99_USA_2005 G R G G W C Y Y M A T Q K R V Q E V R G Y T K G G P G H E E [2640]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2640]

NY99_USA_2005 P Q L V Q S Y G W N I V T M K S G V D V F Y R P S E C C D T [2670]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2670]

NY99_USA_2005 L L C D I G E S S S S A E V E E H R T I R V L E M V E D W L [2700]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2700]

NY99_USA_2005 H R G P R E F C V K V L C P Y M P K V I E K M E L L Q R R Y [2730]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2730]

NY99_USA_2005 G G G L V R N P L S R N S T H E M Y W V S R A S G N V V H S [2760]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2760]

NY99_USA_2005 V N M T S Q V L L G R M E K R T W K G P Q Y E E D V N L G S [2790]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2790]

NY99_USA_2005 G T R A V G K P L L N S D T S K I K N R I E R L R R E Y S S [2820]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2820]

NY99_USA_2005 T W H H D E N H P Y R T W N Y H G S Y D V K P T G S A S S L [2850]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2850]

NY99_USA_2005 V N G V V R L L S K P W D T I T N V T T M A M T D T T P F G [2880]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2880]

NY99_USA_2005 Q Q R V F K E K V D T K A P E P P E G V K Y V L N E T T N W [2910]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2910]

NY99_USA_2005 L W A F L A R E K R P R M C S R E E F I R K V N S N A A L G [2940]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2940]

NY99_USA_2005 A M F E E Q N Q W R S A R E A V E D P K F W E M V D E E R E [2970]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [2970]

NY99_USA_2005 A H L R G E C H T C I Y N M M G K R E K K P G E F G K A K G [3000]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3000]

NY99_USA_2005 S R A I W F M W L G A R F L E F E A L G F L N E D H W L G R [3030]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3030]

NY99_USA_2005 K N S G G G V E G L G L Q K L G Y I L R E V G T R P G G K I [3060]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3060]

NY99_USA_2005 Y A D D T A G W D T R I T R A D L E N E A K V L E L L D G E [3090]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3090]

NY99_USA_2005 H R R L A R A I I E L T Y R H K V V K V M R P A A D G R T V [3120]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3120]

NY99_USA_2005 M D V I S R E D Q R G S G Q V V T Y A L N T F T N L A V Q L [3150]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3150]

NY99_USA_2005 V R M M E G E G V I G P D D V E K L T K G K G P K V R T W L [3180]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3180]

NY99_USA_2005 F E N G E E R L S R M A V S G D D C V V K P L D D R F A T S [3210]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3210]

NY99_USA_2005 L H F L N A M S K V R K D I Q E W K P S T G W Y D W Q Q V P [3240]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3240]

NY99_USA_2005 F C S N H F T E L I M K D G R T L V V P C R G Q D E L V G R [3270]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3270]

NY99_USA_2005 A R I S P G A G W N V R D T A C L A K S Y A Q M W L L L Y F [3300]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3300]

NY99_USA_2005 H R R D L R L M A N A I C S A V P V N W V P T G R T T W S I [3330]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3330]

NY99_USA_2005 H A G G E W M T T E D M L E V W N R V W I E E N E W M E D K [3360]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . [3360]

NY99_USA_2005 T P V E K W S D V P Y S G K R E D I W C G S L I G T R A R A [3390]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . [3390]

NY99_USA_2005 T W A E N I Q V A I N Q V R A I I G D E K Y V D Y M S S L K [3420]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [3420]

NY99_USA_2005 R Y E D T T L V E D T V L * [3434]

ATH002316_Kenya_2012 . . . . . . . . . . . . . . [3434]

Sequence alignment of the WNV polyprotein gene that corresponds to the structural (C, E and M) and non-structural (1, 2A, 2B, 3, 4A, 4B and 5) proteins sequences of the Kenyan WNV isolate (ATH002316) and the reference strain (NC_009942.1). Region sequenced; position 1 to 3434. Study isolate highlighted in red.