Infection, Genetics and Evolution 24 (2014) 116–126

Contents lists available at ScienceDirect

Infection, Genetics and Evolution

journal homepage: www.elsevier .com/locate /meegid

Chikungunya virus adaptation to a mosquito vector correlates with onlyfew point mutations in the viral envelope glycoprotein

http://dx.doi.org/10.1016/j.meegid.2014.03.0151567-1348/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Department of Virology, Arboviruses and InsectVectors, Institut Pasteur, 25-28 Rue du Docteur Roux, 75015 Paris, France. Tel.: +33(0)140613617.

E-mail address: camilo.arias-goeta@pasteur.fr (C. Arias-Goeta).1 Current address: AFFSA LERPAZ, UMR BIPAR, AFSSA, ENVA, UPVM, Maisons-Alfort,

France.

Camilo Arias-Goeta a,b,⇑, Sara Moutailler a,1, Laurence Mousson a, Karima Zouache a,Jean-Michel Thiberge c, Valérie Caro c, François Rougeon d, Anna-Bella Failloux a

a Department of Virology, Arboviruses and Insect Vectors, Institut Pasteur, Paris, Franceb Cellule Pasteur UPMC, Université Pierre et Marie Curie, Paris, Francec Department of Infection and Epidemiology, Genotyping of Pathogens and Public Health, Institut Pasteur, Paris, Franced URA 2581, Genetic and Molecular Interactions Cell-eucaryote, Institut Pasteur, Paris, France

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 December 2013Received in revised form 27 February 2014Accepted 17 March 2014Available online 26 March 2014

Keywords:Host alternationArbovirus evolutionGenome stabilityVector adaptation

Like most arthropod-borne viruses (arboviruses), chikungunya virus (CHIKV) is a RNA virus maintained innature in an alternating cycle of replication between invertebrate and vertebrate hosts. It has beenassumed that host alternation restricts arbovirus genome evolution and imposes fitness trade-offs.Despite their slower rates of evolution, arboviruses still have the capacity to produce variants capableto exploit new environments.

To test whether the evolution of the newly emerged epidemic variant of CHIKV (E1-226V) isconstrained by host alternation, the virus was alternately-passaged in hamster-derived BHK-21 cellsand Aedes aegypti-derived Aag-2 cells. It was also serially-passaged in BHK-21 or Aag-2 cells to promoteadaptation to one cell type and presumably, fitness cost in the bypassed cell type. After 30 passages,obtained CHIKV strains were genetically and phenotypically characterized using in vitro and in vivosystems.

Serially- and alternately-passaged strains can be distinguished by amino-acid substitutions in the E2glycoprotein, responsible for receptor binding. Two substitutions at positions E2-64 and E2-208 onlylower the dissemination of the variant E1-226V in Ae. aegypti. These amino-acid changes in the E2 glyco-protein might affect viral infectivity by altering the interaction between CHIKV E1-226V and the cellularreceptor on the midgut epithelial cells in Ae. aegypti but not in Aedes albopictus.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

Mechanisms leading to virus emergence are complex combin-ing both ecological and genetic factors (Cleaveland et al., 2007).Even though they are small in size (�104 base pairs in length)and contain a small number of genes, RNA viruses are character-ized by high rates of mutations (10�4 errors/nucleotide/round ofreplication) (Holland et al., 1982; Steinhauer and Holland, 1987;Domingo and Holland, 1997; Drake and Holland, 1999). This isdue to a lack of proof-reading repair activities associated withRNA-dependent RNA-polymerase (RdRp) (Steinhauer et al., 1992).As a consequence of their rapid replication cycle and their large

population size, arboviruses can exist as mixed populations ofgenomic variants that are closely related but not genetically iden-tical (Lin et al., 2004; Fitzpatrick et al., 2010; Lauring and Andino,2010; Coffey et al., 2011; Domingo et al., 2012). The resultingquasispecies can confer phenotypic plasticity and subsequentadaptability to new environments (Domingo and Holland, 1997;Eigen, 1996; Pfeiffer and Kirkegaard, 2005). Nevertheless, strongpurifying selection seems to dominate the evolution of arboviruses(Jerzak et al., 2005). It has been postulated that this stability mayresult from host alternation cycling, since arboviruses must repli-cate in disparate hosts (vertebrate and invertebrate) suggestingthat the only mutations fixed are neutral or beneficial in both hosts(Ciota and Kramer, 2010; Novella et al., 2011).

In vitro and in vivo experimental evolution approaches havebeen widely used to test this hypothesis. Viral evolution has beencompared between viruses sequentially passaged in one host andviruses passaged in alternation between the two hosts. Even ifin vitro passaged viruses undergo artificial adaptation to host cell

C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126 117

molecules, these experiments revealed that fitness constraints dif-fer in vertebrate and invertebrate cells. And, viral fitness in bothcell types is not always limited by host alternation (Weaveret al., 1999; Novella et al., 1999; Greene et al., 2005; Vasilakiset al., 2009). However, alternating passages of virus can be neces-sary to maintain viral genome integrity as well as virulence(Moutailler et al., 2011). In vivo studies with VEEV (Coffey et al.,2008) and WNV (Deardorff et al., 2011) showed convergent resultssince viruses serially-passaged in vertebrates or mosquitoes led toviruses specialized to each host, whereas viruses passaged in alter-nation did not reflect necessarily an increased fitness in each host.Based on consensus sequences, a greater number of mutationshave been found invariably in serially-passaged viruses (Greeneet al., 2005; Moutailler et al., 2011) as well as in alternately-passaged viruses (Novella et al., 1999; Vasilakis et al., 2009)depending on the cell culture system used. Furthermore, comparedto viruses selected using in vitro system, in vivo adapted virusesshowed fewer nucleotide changes in consensus sequencessuggesting that this environment is less permissive for mutationsto be fixed rapidly (Coffey et al., 2008). Moreover, genetic diversityis not necessarily a requisite for increased viral fitness in thespecialized host (Coffey and Vignuzzi, 2011; Deardorff et al., 2011).

Viral emergence begins with a stochastic process that involvesthe transmission of a preexisting viral strain into a new host spe-cies, followed by adaptation to the new host (Elena et al., 2011).The potential of arboviruses to expand and emerge in naïve envi-ronments and/or to adapt to novel hosts has been widely docu-mented for dengue viruses (DENV) (Wang et al., 2000; Moncayoet al., 2004), West Nile virus (WNV) (Moudy et al., 2007) andVenezuelan equine encephalitis virus (VEEV) (Brault et al., 2004).The last outbreak of CHIKV in 2005–2007 in the Indian Ocean re-gion is also a good example: a single amino-acid change from analanine to a valine at position 226 of the E1 viral glycoprotein(E1-226V) was associated with an enhanced dissemination andtransmission by the unusual vector Aedes albopictus (Tsetsarkinet al., 2007; Vazeille et al., 2007). Until this event, the main vectorof CHIKV was the urban mosquito, Aedes aegypti. However, duringthe outbreak on La Réunion, Ae. albopictus was the only vector in-volved (Bagny et al., 2009), suggesting a specific CHIKV adaptationto this mosquito species. This adaptation seems to have occurredindependently at least three times during outbreaks in the IndianOcean region, the Indian subcontinent, and Central Africa(de Lamballerie et al., 2008; Tsetsarkin and Weaver, 2011). Thus,a single mutation can have a profound effect on transmission bya particular host despite slow viral rates of evolution. Nevertheless,one question remains: can the effect of this mutation be compro-mised when the transmission cycle repeatedly involves the urbanand anthropophilic mosquito Ae. aegypti, the historical vector ofCHIKV (Powers and Logue, 2007).

In this study, we examined the consequences of host alternationon the evolution of the newly emerged epidemic variant CHIKVE1-226V. We used hamster cells (BHK-21) and Ae. aegypti cells(Aag-2) for serial and alternate passages. Viral fitness was assessedthrough determining the replicative capacity of viral variantsin vitro cultured cells or in vivo host tissues. Our results supportthe hypothesis that host alternation restricts CHIKV evolutionsince the alternately-passaged virus showed decreased geneticdiversity than the serially-passaged viruses without affectingvirulence in mice and dissemination in mosquitoes. Moreover,selection by serial passages in one cell type leads to viruses special-ized to the corresponding host. Two amino-acid substitutions inthe E2 glycoprotein at positions E2-64 and E2-208 affect dissemi-nation of the variant E1-226V in Ae. aegypti and not in Ae. albopictus.We suggest that amino-acid changes in this region of E2 proteinmight disrupt interactions between the virus and the receptors pres-ent on the midgut cells of Ae. aegypti only. Nevertheless, these substi-

tutions are not detected in currently circulating CHIKV strainsworldwide.

2. Materials and methods

2.1. Ethics statement

Mice used in this study were housed in the Institut Pasteur ani-mal facilities accredited by the French Ministry of Agriculture forperforming experiments on live rodents. Work on animals was per-formed in appliance with French and European regulations on careand protection of laboratory animals (EC Directive 2010/63, FrenchLaw 2013-118, February 6th, 2013). Protocols were approved bythe veterinary staff of the Institut Pasteur animal facility and wereperformed in compliance with the NIH Animal Welfare Insurance#A5476-01 issued on 31/07/2012.

2.2. Viruses

Chikungunya strain CHIKV 06.21 (E1-226V) isolated in LaRéunion in November 2005, from a newborn male presenting men-ingo-encephalitis symptoms, was used as the parental strain (i.e.,the starting point for experimental in vitro selection). This straincontaining the A226V mutation was provided by the FrenchNational Reference Center for arboviruses and has been entirelysequenced (GenBank No.: AM258992). Stocks were produced onAe. albopictus C6/36 cells after three passages, then harvested andstored at �80 �C until use. Viral titer estimated by serial tenfolddilutions on Vero cells was 109 plaque forming units (pfu)/mL.Moreover, the other viral strains obtained after 30 passagesin vitro (see below) were produced at high titers (Table S1):P30Alt at 107.9 pfu/mL, P30BHK21 at 108.7 pfu/mL and P30Aag2 at108.1 pfu/mL. Biological clones selected in vitro (see below) werealso produced on Ae. albopictus C3/36 cells: E2-7N at 107.8

pfu/mL, E2-7S at 107.8 pfu/mL, E2-60D at 107.9 pfu/mL, E2-60N at108.0 pfu/mL, E2-64W/208E at 107.2 pfu/mL, E2-64R/208E at108.7 pfu/mL and E2-64W/208K at 108.7 pfu/mL.

2.3. Cells

Four cell types were used: (i) Aag-2, an Ae. aegypti cell lineage ofembryonic origin (Peleg, 1968) commonly used as a model formosquito immune studies (Fallon and Sun, 2001), (ii) C6/36,Ae. albopictus cells originally established from mosquito larvaehomogenates (Igarashi, 1978) are defective in typical siRNAs, thehallmark of exogenous RNAi mediated antiviral immunity(Brackney et al., 2010), (iii) BHK-21, a hamster cell line derivedfrom kidney fibroblasts (ATCC�, Manassas, USA) (MacPhersonand Stoker, 1962) are recognized to have a defect in interferonproduction (Otsuki et al., 1979), and (iv) Vero cells isolated fromkidney epithelial cells of an African green monkey (ATCC�,Manassas, USA) (Yasumura and Kawakita, 1963) are interferon-deficient (Desmyter et al., 1968) and commonly used for viral titra-tion. Insect cell lines were incubated at 28 �C: C6/36 (Ae. albopictus)cells were maintained in Leibowitz L-15 medium with 1%non-essential amino-acids (Invitrogen), 10% fetal bovine serum(FBS) and 1% penicillin/streptomycin (P/S) (Invitrogen) and Aag-2(Ae. aegypti) cells in Schneider Drosophila medium, 10% FBS and1% P/S. Mammalian cell lines were incubated at 37 �C with 5%CO2: BHK-21 and Vero cells were maintained in Dulbecco’sModified Eagle medium (DMEM) with 10% FBS and 1% P/S.

2.4. Mosquitoes

In addition to Ae. aegypti, Ae. albopictus was also tested to mea-sure the fitness of serially- and alternately-passaged viral strains.

Table 1Amino-acid substitutions observed in the CHIKV E1-226V genome.

nsP1 nsP3 E2

G230R M314V G117R M424I N7S D60N W64R E208K

Parental 230G 314M 117G 424M 7N 60D 64W 208EP30Alt X X X X XP30BHK21 X X X X XP30Aag2 X

Changes in the serially- and alternately-passaged strains (P30Alt, P30BHK21 andP30Aag2) were recorded in reference to the parental strain (E1-226V). X means thatan amino-acid substitution is observed.

118 C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126

Aedes aegypti Petite Terre (AAPT) from Mayotte collected in Decem-ber 2006 and Aedes albopictus Providence (ALPROV) from LaRéunion collected in March 2007 (Martin et al., 2010) were usedfor experimental infections. The F13 and F7 generation respectivelywere used for AAPT and ALPROV. Eggs were hatched in water andlarvae reared in pans containing one yeast tablet per liter ofdechlorinated tap water. Adults were maintained at 28 ± 1 �C,80% relative humidity and a light:dark cycle of 16 h:8 h. A constantsupply of 10% sucrose was provided to adults. Females were fedthree times a week on mice (OF1 mice from Charles River labora-tories, France).

2.5. Serial/alternate experimental passages of viruses

Parental CHIKV strain (E1-226V) was serially passaged 30 timesin BHK-21 or Aag-2 cells, or alternately passaged between BHK-21and Aag-2 cells (i.e., 15 passages in BHK-21 and 15 passages inAag-2 with the 30th passage ending on BHK-21 cells). Confluentcell monolayers prepared in 75 cm2 flasks were infected at a mul-tiplicity of infection (MOI) of 0.1 for 1 h in appropriate conditions.After adsorption, the inoculum was removed, cell monolayers werewashed with the medium and 13 mL of fresh medium with 2% FBSwere added. Cells were incubated in appropriate conditions untilsupernatant titers reached a plateau at 48 h post-infection (pi).At each passage, supernatants were harvested, stored in aliquotsat �80 �C and titrated on Vero cells. The viruses obtained at the30th passage (alternately-passaged P30Alt, serially-passaged onmammalian cells P30BHK21 and serially-passaged on mosquitocells P30Aag2) were titrated and used for in vitro and in vivo fitnessexperiments. Each passage corresponds to the transfer of �1–2 106

viral particles (at a MOI of 0.1, 1–2 106 viruses infect 1–2 107 cellsin a 75 cm2 flask), which can be considered large enough to mini-mize the effect of bottleneck on the entire viral population and lowenough to minimize the effect of defective interfering (DI)particles.

2.6. In vitro kinetics of viral growth

In vitro kinetics of the parental E1-226V, P30Alt, P30BHK21 andP30Aag2 strains was defined by measuring kinetics of viral growthon cells. Growth curves of the parental E1-226V, P30Alt, P30BHK21and P30Aag2 were produced by infecting BHK-21, Aag-2, andC6/36 cells at a MOI of 0.1 for 1 h in appropriate conditions.Supernatants were collected at 2, 4, 6, 8, 10, 12, 24, 48, and72 h pi and titrated on Vero cells. Viral titer was expressed in pfu/mL.

2.7. Mice infection and viral replication

Replication capacity of the parental E1-226V, P30Alt, P30BHK21and P30Aag2 strains was determined by measuring survival of IFN-a/bR�/� (129s/v) mice after viral inoculation (Couderc et al., 2008).Mice provided by the Institut Pasteur were bred according to theInstitut Pasteur guidelines for laboratory animal husbandry andkept in level-3 isolators. Four to six-week-old females were in-fected intradermally with 20 pfu of each CHIKV strain. The controlmice were inoculated with 1X PBS. Batches of ten mice were usedfor mortality studies. The viral titer (i.e., number of infectious viralparticles) in liver, spleen, muscle and brain tissues was determinedby titration on Vero cells of extracts obtained from ground organs.

2.8. Mosquito infection and viral dissemination

Replication of the parental E1-226V, P30Alt, P30BHK21 andP30Aag2 strains in mosquitoes was defined by analyzing individu-als able to disseminate the virus in mosquito internal organs afteringestion of the infectious blood-meal. Disseminated infection rate

(DIR) corresponding to the proportion of mosquitoes with virus de-tected in the head, indicative of efficient viral dissemination, andviral loads describing the intensity of viral dissemination, werecompared. Infection assays were performed with 1-week-oldAe. aegypti (AAPT) and Ae. albopictus (ALPROV) females in a BSL-3laboratory. Two batches of 60 individuals starved for 24 h priorto infection were allowed to feed for 15 min through a pig intestinemembrane covering the base of a glass feeder containing the infec-tious meal maintained at 37 �C. The infectious blood-meal wascomposed of 2 mL of washed erythrocytes isolated from rabbitarterial blood collected before infection and 1 mL of viral suspen-sion of each virus: E1-226V, P30Alt, P30BHK21 or P30Aag2.Adenosine triphosphate was added as a phagostimulant at a finalconcentration of 5 � 10�3 M. The final concentration of viralsuspension was 106.5 pfu/mL. The entire feeding period lasted15 min without any significant variation detected in viral titersof blood-meals. After feeding, engorged females were sorted onice and placed in cardboard containers. Then they were fed with10% sucrose at 28 ± 1 �C, 80% of relative humidity and a light:darkcycle of 16 h:8 h.

Biological clones harboring the following substitutions – E2-7N,E2-7S, E2-60D, E2-60N, E2-64W/208E, E2-64R/208E or E2-64W/208K (for more details, see Table 1) – produced by plaque purifica-tion on Vero cells (see below) were used to test their ability to dis-seminate successfully in internal organs or tissues (i.e., salivaryglands, head.) of Ae. aegypti and Ae. albopictus mosquitoes. 1 mLof viral suspension containing one biological clone was added tothe blood-meal as described above.

To evaluate viral dissemination, mosquito heads were ground in200 lL of DMEM at day 7 pi. Samples were added to a monolayer ofVero cells to detect infectious particles using the plaque assaytechnique.

2.9. Production of biological clones

Biological clones of serially- or alternately-passaged virusesharboring the following substitutions – E2-7N, E2-7S, E2-60D,E2-60N, E2-64W/208E, E2-64R/208E or E2-64W/208K (Table 1) –were isolated using the plaque assay technique on Vero cells. Eachagarose plug that contained an individual clone was dissolved in100 lL of DMEM. The biological clones were amplified in Aag-2or BHK-21 cells depending on the type of cells used for the passage.

2.10. Viral titration by plaque assay

Six-well-plates containing confluent monolayers of Vero cellswere infected with tenfold dilutions of viral supernatants. Cellswere incubated at 37 �C with 5% CO2 under an overlay consistingof DMEM (1X) with 2% FBS, 1% L-Glutamine, 1% agarose and 1%penicillin/streptomycin/amphotericin (Invitrogen). Three daysafter, the agar plugs were removed and the cell monolayers werefixed with a solution of 10% formalin, 0.2% crystal violet and 10%ethanol for 30 min.

Fig. 1. Experimental strategy to test CHIKV E1-226V evolution in vitro. The parentalstrain E1-226V was serially passaged 30 times in BHK-21 or Aag-2 cells, oralternately passaged between BHK-21 and Aag-2 cells using a MOI of 0.1 (pfu/cell).Genetic changes were detected by comparing the consensus sequence of the entiregenome of variants obtained at the 30th passage, and by sequencing the E2 gene often biological clones isolated every five passages. The fitness of the parental strain,P30Alt, P30BHK21 and P30Aag2 was tested in vitro on Aag-2, C6/36 and BHK-21 celllines and in vivo after inoculation in mice and oral infection of two mosquito species(Ae. aegypti and Ae. albopictus).

C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126 119

2.11. RT-PCR amplification and genome sequencing

Total RNA was extracted from the parental E1-226V and thesupernatants of the 30th passage of P30Alt, P30BHK21 andP30Aag2. Extraction was carried out using NucleoSpin II RNA kit(Macherey Nagel) according to the manufacturer’s instruction.Sequencing of the viral genome was performed using primersdesigned to obtain 700 bp RT-PCR amplicons covering the entiregenome. Amplicons generated using the Titan One Tube kit (Roche)presented an overlap of 100 bp between contiguous fragments(Table S2). The one-step RT-PCR reaction was performed in a vol-ume of 50 lL containing 5 lL RNA template, 29.5 lL ddH2O, 1 lLdNTP (10 mM), 2.5 lL DTT (100 mM), 10 lL RT-PCR buffer (5X),0.5 lL sense oligonucleotide (50 lM), 0.5 lL anti-sense oligonu-cleotide (50 lM), 0.5 lL RNase inhibitor (5 U/lL) and 0.5 lL Titanenzyme mix. The amplification program was performed as follows:reverse transcription at 50 �C for 30 min, an inactivation of RTenzyme step at 94 �C for 2 min, followed by 35 cycles of 94 �C15 s, 60 �C 30 s, 72 �C 1 min 30 s, and a final step at 72 �C for10 min. PCR products were purified using the NucleoFast kit(Macherey Nagel) as specified by the manufacturer. Sequencingwas carried out using the ABI Prism BigDye Terminator CycleSequencing Ready Reaction kit version 3.1 (Applied Biosystems).The sequencing reaction was performed in a volume of 10 lL con-taining 1 lL PCR product template, 5.2 lL ddH2O, 2 lL sequencingbuffer (5X), 1 lL oligonucleotide (4 lM) and 0.8 lL ABI Prismsolution version 3.1. The sequencing program was performed asfollows: 96 �C 1 min followed by 30 cycles of 96 �C 10 s, 50 �C5 s, 60 �C 4 min. Sequence chromatograms for both strands wereobtained using an automated sequence analyzer ABI3730XL(Applied Biosystems). Sequence analysis and alignment were per-formed using the software BioNumerics v 5.1 (Applied-Maths).

2.12. Genetic diversity over the course of passages

Viral supernatants, collected every five passages (5th to 30thpassage) of serially and alternately-passaged viruses, were usedto isolate biological clones. Six-well-plates containing confluentmonolayers of Vero cells were infected with tenfold dilutions ofviral supernatants. Cells were incubated for 3 days at 37 �C and5% CO2 under an overlay consisting of DMEM (1X) with 2% FBS,1% L-Glutamine, 1% agarose and 1% penicillin/streptomycin/amphotericin (Invitrogen). Ten lytic plaques were localized and re-moved by suction using a pipette for each supernatant tested. Eachagarose plug that contained an individual clone was dissolvedovernight at +4 �C in 50 lL of DMEM. Total RNA from supernatantswas extracted using NucleoSpin 96 RNA kit (Macherey Nagel)according to the manufacturer’s instruction. To evaluate geneticdiversity between the different biological clones isolated fromthe same supernatant, a RT-PCR targeting the viral E2 gene wasconducted; the choice of E2 gene was based on genome sequenceof in vitro selected viruses (for more details, see Table 1). Theone-step RT-PCR reaction was performed using the Titan One Tubekit (Roche) as described above. Fragments 17 and 18, covering theentire E2 sequence were generated (Table S2). The sequencingreaction was conducted using the ABI Prism BigDye TerminatorCycle Sequencing Ready Reaction kit version 3.1 (Applied Biosystems)as described above.

2.13. Statistical analysis

All statistical analyses were performed using the Stata soft-ware (StataCorp LP, Texas, and USA). To detect significant differ-ences in viral titers among experimental groups, comparisonswere done using the Kruskal–Wallis test or the Mann–Whitneytest. The Chi square test was used to detect significant differences

in disseminated infection rates among groups. Significance levelsfor multiple testing were corrected using sequential Bonferroni’sprocedures (Holm, 1979). Mice survival was described using Kap-lan–Meier survival curves, which were compared with the logrank test.

3. Results

The strategy adopted to examine the role of alternating replica-tion on CHIKV evolution is described in Fig. 1.

3.1. Viral replication in vitro

To determine if serial passages of CHIKV led to an increasedcapacity to replicate in the cell line used for passages or decreasedreplication capacity in the bypassed cell line, we examined the rep-lication kinetics of the parental E1-226V, P30Alt, P30BHK21, andP30Aag2 viruses in BHK-21, Aag-2 and C6/36 cell lines. Two repli-cates were performed for each cell-virus pairing.

In BHK-21 cells (Fig. 2A), the serially-passaged viruses in BHK-21 cells (P30BHK21) and the alternately-passaged (P30Alt) viruses,presented the highest viral titers compared to the parental strainfrom 6 h to 48 h pi. From 48 h pi a cytopathogenic effect was ob-served only in cells infected with the parental E1-226V, P30Alt,and P30BHK21 strains, leading to a decrease in viral titers. This ef-fect was not observed in cells infected with the P30Aag2 strain,resulting in a higher viral titer at 72 h pi when compared to theother viral strains. In Aag-2 cells (Fig. 2B), no difference in viral ti-ters was observed between the parental E1-226V, P30BHK21, andP30Alt. In Ae. albopictus C6/36 cell line, defective in antiviral im-mune responses, high viral loads were produced for the parentalE1-226V, P30Alt, and P30BHK21 strains (Fig. 2C). Interestingly,the serially-passaged viruses in Aag-2 cells (P30Aag2) exhibit thelowest viral titers in all cell lines including the Ae. albopictus-de-rived C6/36 cells. Nevertheless, it seems to depend on cells usedfor experimental selection as the serially-passaged in BHK-21 cells(P30BHK21) strain presented high capacities to replicate in all cell

Fig. 2. Growth curves of CHIKV strains in three cell lines. (A) Baby hamster kidney BHK-21 cells, (B) Ae. aegypti Aag-2 cells and (C) Ae. albopictus C6/36 cells were infected withthe parental E1-226V (black solid line), the P30Alt (black dotted line), the P30BHK21 (grey solid line) and the P30Aag2 (grey dotted line) strains at a MOI of 0.1. After 1 h ofadsorption, the inoculum was removed and cells were washed. Then, medium with 2% FBS was added and cells were incubated at 28 �C for mosquito cells and 37 �C with 5%CO2 for mammalian cells. Supernatants were collected at 2, 4, 6, 8, 10, 12, 24, 48 and 72 h post-infection. The number of infectious viral particles was determined by plaqueassay on Vero cells. Two trials were performed for each cell-virus pairing. Error bars show standard deviations.

120 C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126

Fig. 3. Survival of mice inoculated with CHIKV strains. Batches of ten IFN-a/bR�/� (129s/v) mice were infected intradermally with 20 pfu of each CHIKV strain: parental E1-226V, P30Alt, P30BHK21, and P30Aag2. The control mice were inoculated with PBS 1X. Mice survival rate was recorded over 7 days post-infection.

C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126 121

lines. The two replicates were comparable as error bars presentedlimited extent.

3.2. Viral replication in mice

To evaluate the virulence of the parental strain, the serially-pas-saged and the alternately-passaged CHIKV strains, IFN-a/bR�/�

(129s/v) female mice were infected intradermally with each viralstrain. Mouse survival rate was recorded over 7 days pi (Fig. 3).The control mice, inoculated with PBS, survived until day 7 pi. Allother batches of mice died between day 3 and day 6 pi with differ-ent patterns (log-rank test: p = 0.002). When compared to theparental strain, the P30Alt strain behaved similarly (log-rank test:p > 0.05) and the P30BHK21 strain died later than the otherinoculated mice.

To assess if mouse mortality was associated with increased viralloads, the viral titer (i.e., number of infectious viral particles) inliver, spleen, muscle and brain tissues was determined just at thetime of mouse death (Fig. 4). Viral loads varied according to organsand the virus. The highest viral titers were detected in the liver.Compared to the parental strain, the P30Aag2 strain showed thelowest viral titers in all examined organs (Mann–Whitney test:p < 0.05). Interestingly, the parental strain causing the highestmortality rates (Fig. 3) did not produce the highest viral loads

Fig. 4. Viral titers in tissues of mice inoculated with CHIKV strains. Batches of ten IFN-a/bOrgans (liver, brain, spleen and muscle) were dissected immediately after mouse death acells. Each boxplot represents median and range values of viral titers in 10 organs. ⁄⁄⁄p 6 0Mann–Whitney test.

suggesting that mouse mortality was not correlated with high viraltiters in organs.

3.3. Viral dissemination in mosquitoes

To study viral dissemination within mosquitoes, Ae. aegypti(AAPT) and Ae. albopictus (ALPROV) were orally infected with eachviral strain: the parental E1-226V, the serially-passaged(P30BHK21 and P30Aag2) and the alternately-passaged P30Altstrains. Viral titers in mosquito heads (i.e., describing intensity ofviral dissemination) and disseminated infection rates (i.e., corre-sponding to the proportion of mosquitoes with virus detected inthe head) were determined at day 7 pi (Arias-Goeta et al., 2013).Two trials were performed and ten individuals were tested for eachmosquito-virus pairing.

We first considered the disseminated infection rates corre-sponding to the proportion of mosquitoes able to ensure an effi-cient viral dissemination (Fig. 5A). Compared to the parentalstrain, the P30BHK21 strain showed the lowest DIR when infectingAe. aegypti AAPT (Chi square test: p < 0.05) and the P30Aag2 strainpresented the lowest DIR in Ae. albopictus ALPROV (Chi square test:p < 0.05). When comparing DIR between the two mosquito speciesafter infection with each viral strain, significant differences werefound suggesting that Ae. aegypti AAPT and Ae. albopictus ALPROV

R�/� (129s/v) mice were inoculated with 20 pfu of CHIKV via the intradermal route.nd the amount of infectious viral particles was determined by plaque assay on Vero.001; ⁄⁄p 6 0.01; ⁄p 6 0.05; NS, non-significant compared to the parental strain after

Fig. 5. CHIKV dissemination (A) and viral loads (B) in Ae. aegypti AAPT and Ae.albopictus ALPROV. Mosquitoes were orally infected with a blood-meal containing106.5 pfu/mL of each CHIKV strain: parental E1-226V, P30Alt, P30BHK21 andP30Aag2. To measure viral dissemination, viral titers were determined in twobatches of ten mosquito heads at day 7 post-infection. ⁄⁄⁄p 6 0.001; ⁄⁄p 6 0.01;⁄p 6 0.05; NS, non-significant compared to the parental strain after Chi square test(A) or Mann–Whitney test (B).

122 C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126

do not disseminate the different viral strains with the same effi-ciency (Chi square test: p < 0.05).

To describe the intensity of viral dissemination, viral loads in in-fected mosquito heads were examined for mosquitoes that havedisseminated the virus (Fig. 5B). For Ae. aegypti AAPT, no differencewas detected when comparing with the parental strain (Mann–Whitney test: p > 0.50). For Ae. albopictus ALPROV, a significant dif-ference in viral loads was found for the P30BHK21 and theP30Aag2 strains compared to the parental strain (Mann–Whitneytest: p < 0.05).

To sum up, the four viral strains disseminated weakly in Ae.aegypti AAPT, except for the P30Aag2 strain that disseminatedbetter in the host whose cell line was used for in vitro selectionby serial passages. This may suggest a positive selection of theP30Aag2 strain by Ae. aegypti-derived Aag-2 cells.

3.4. Sequence analysis of the entire genome

Consensus sequences of the entire genomes of the parentalstrain, the serially-passaged and the alternately-passaged CHIKVstrains, were compared. CHIKV-P30Alt showed five substitutionswith amino-acid change (Table 1): two in nsP1 (G230R andM314V), two in nsP3 (G117R and M424I) and one in the E2 glyco-protein (D60N). The P30BHK21 strain showed five substitutions:two substitutions in nsP1 (G230R and M314V), one substitution

in nsP3 (G117R) and two substitutions in the E2 glycoprotein(W64R and E208K). CHIKV-P30Aag2 showed only one amino-acidsubstitution in the E2 glycoprotein (N7S). Three substitutions(nsP1-G230R, nsP1-M314V and nsP3-G117R) were identical inthe P30BHK21 and P30Alt strains. This may result from an adapta-tion of the parental strain to molecules only present in BHK-21cells. Interestingly, serially- and alternately-passaged strains couldbe distinguished by four amino-acid substitutions in the E2 glyco-protein: N7S, D60N, W64R, and E208R. Thus sequences of E2 genewere generated to distinguish individual biological clones presentin the four viral strains.

3.5. Sequence analysis of the E2 gene of biological clones

Every five passages, ten biological clones were isolated fromsupernatants of serially- and alternately-passaged CHIKV strains.Comparisons of E2 gene sequences of the ten biological clones ob-tained from each supernatant showed different proportions of thefour substitutions previously highlighted (Table 2). When com-pared to the parental E1-226V strain, the alternately-passagedviruses presenting one amino-acid substitution from an asparticacid to an asparagine at position E2-60, started to be detected afterthe 15th alternate passage (60%). For the Aag-2 serially-passagedviruses, one substitution at position E2-7 from an asparagine to aserine was observed at high frequency after the 20th passage(60%). Lastly, for the BHK-21 serially-passaged viruses, two ami-no-acid substitutions were observed. The substitution at positionE2-64 from a tryptophan to an arginine was detected from the10th passage (30%) and present in the following passages at vari-able proportions from 20% to 50%. Moreover, the substitution atposition E2-208 from a glutamic acid to a lysine was detected fromthe 10th passage (40%) and present in most clones examined fromthe 15th passage up to the 30th passage. Surprisingly, the biologi-cal clones with the E2-208K substitution did not present the E2-64R substitution, and conversely.

3.6. Dissemination in mosquitoes of viral clones presenting differentsubstitutions in E2 gene

To define if substitutions in the E2 gene play a key role in viraldissemination within mosquitoes, and consequently have an epi-static effect on the E1-A226V substitution, Ae. aegypti AAPT andAe. albopictus ALPROV were orally infected with a biological clonediffering from the wild-type variant by one single mutation. Cloneswere produced for each type of passage (P30Alt, P30BHK21, andP30Aag2). Dissemination rates and head viral loads were estimatedin ten mosquitoes and then compared.

When considering the disseminated infection rate (Fig. 6A), Ae.aegypti AAPT infected with the parental strain were able to dissem-inate the virus with infectious viral particles detected in 70% ofmosquitoes. As expected, Ae. albopictus ALPROV was highly effi-cient in ensuring the dissemination of the parental strain (100%).When comparing DIR obtained from infections with the differentbiological clones (Fig. 6A), significant differences were found withAe. aegypti AAPT and Ae. albopictus ALPROV (Chi square test:p < 0.05). Additionally, significant lower DIR were found in Ae. ae-gypti AAPT for E2-60N, E2-64R/208E and E2-64W/208K when com-pared to the wild-type variant, respectively E2-60D, E2-64W/208Eor E2-7N (Chi square test: p < 0.05). Surprisingly, the substitutionin E2-64/208 did not affect DIR in Ae. albopictus ALPROV (Chisquare test: p > 0.05). Thus the substitution in E2-64/208 loweredviral dissemination in Ae. aegypti AAPT whereas it had no effectin Ae. albopictus ALPROV. When examining viral titers in infectedmosquito heads (Fig. 6B), no significant differences were detectedamong biological clones isolated from a given type of passage(Kruskal–Wallis: p < 0.05).

Table 2Amino-acid changes observed in the E2 glycoprotein of CHIKV biological clones.

Passage Clone Alternate Serial Aag2 Serial BHK21

D60 N7 W64 E208

P5 1 – – – –2 – – – –3 – – – –4 – – – –5 – – – –6 – – – –7 – – – –8 – – – –9 – – – –

10 – – – –

P10 1 – – – K2 – – – –3 – – – K4 – – R –5 – – – K6 – – R –7 – – – K8 – – – –9 – – R –

10 – – – –

P15 1 – – – K2 – – R –3 – – – K4 N – – K5 N – – K6 N – R –7 N – R –8 – – R –9 N – – K

10 N – – K

P20 1 N S – K2 N – – K3 N S – K4 N – R –5 N S – K6 N S – K7 N – – K8 N S – K9 N S – K

10 N – – K

P25 1 N – – K2 N – – K3 N S – K4 N – R –5 N S – K6 N S – K7 N S R –8 N S – K9 N – R –

10 N S – K

P30 1 N S R –2 N S R –3 N S – K4 N S – K5 N S – K6 N S R –7 N S – K8 N – – K9 N S R –

10 N S R –

Every five passages, changes in ten biological clones isolated from the serially- andalternately-passaged strains were recorded in reference to the parental strain (E1-226V). D, aspartic acid; E, glutamic acid; K, lysine; N, asparagine; R, arginine; S,serine; W, tryptophan.

C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126 123

4. Discussion

Since 2005, the E1-226V variant of CHIKV has emerged inde-pendently on at least three occasions during outbreaks in the In-dian Ocean region, the Indian subcontinent, and Central Africa

(de Lamballerie et al., 2008; Tsetsarkin and Weaver, 2011). Vectorcompetence studies revealed that viruses harboring the E1-A226Vsubstitution were better disseminated and transmitted by the unu-sual vector Ae. albopictus (Tsetsarkin et al., 2007; Vazeille et al.,2007). In our study, we tested whether the evolution of the newlyemerged variant of CHIKV was restricted by host alternation mim-icked by an in vitro system comprising an Ae. aegypti cell line and ahamster cell line. Our results show that submitting the parentalE1-226V to alternate cycling affected only slightly its replicativecapacity. However, serial passages on mammalian BHK-21 cellsor mosquito Aag-2 cells allowed selection of clones harboringnew mutations in the E2 gene. Two substitutions in the E2 glyco-protein at positions E2-64 and E2-208 restricted the ability of theE1-226V variant to adapt to Ae. aegypti without any effect on Ae.albopictus. These substitutions in the E2 glycoprotein, located in adomain that interacts with cell receptors (Tsetsarkin and Weaver,2011) are rapidly fixed in the viral population.

After 30 alternate passages between mosquito (Aag-2) andmammalian (BHK-21) cells, slight replicative gains were observedin the BHK-21 cell line (Fig. 2A) and the RNAi-deficient C6/36 cellline (Fig. 2C). An increased viral replication was also detected in li-ver and brain tissues of infected mice (Fig. 4) but was unlikely tocause mouse mortality. No difference in the intensity of viral dis-semination in mosquitoes (estimated by titrating virus in the mos-quito head) was observed between the parental and the P30Altstrains. Host alternation reproduced using an in vitro system didnot alter the characteristics of the parental E1-226V strain. In ourstudy, we considered viral replicative capacity in cultured cells asa proxy of fitness (Quinones-Mateu and Arts, 2006). However otherfactors such as cellular tropism and immune escape should be con-sidered as important components of fitness in hosts. This could ex-plain partially the discrepancies between in vitro and in vivosystems (Domingo and Holland, 1997).

The serially-passaged P30BHK21 on mammalian cells wasweakly able to disseminate within Ae. aegypti mosquitoes, support-ing the hypothesis that adaptation to a single host results in fitnesstrade-offs in the bypassed host. Interestingly, this pattern was notobserved in Ae. albopictus (Fig. 5A). On the other hand, the serially-passaged P30Aag2 on mosquito cells disseminated efficiently inboth mosquitoes, Ae. aegypti and Ae. albopictus (Fig. 5), and had re-duced fitness in mice (Fig. 4). However, the P30Aag2 strain showedlow replication fitness in the three cell lines (Fig. 2) suggesting thatAag-2 cells, used for experimental in vitro selection and presentingefficient antiviral immune responses, may restrict viral evolution.Host specialization through serial passages of an arbovirus in a sin-gle host can result in adaptation to this host and in fitness cost inthe bypassed host (Weaver et al., 1999; Cooper and Scott, 2001;Greene et al., 2005; Vasilakis et al., 2009; Coffey et al., 2008). Nev-ertheless, opposite conclusions can also be obtained (Ciota et al.,2008, 2009; Deardorff et al., 2011). It is important to note thatour experimental design uses cell cultures as ‘‘hosts’’. However,cells are homogeneous environments without any tissue structurepresenting conditions where selection pressures are constant andunidirectional. It should be noted that experiments of virus evolu-tion using in vitro systems might select viruses more adapted tofeatures of cell cultures such as temperature (Novella et al.,1999; Clarke et al., 1993), DI particles or overexpression of heparinsulfate (Klimstra et al., 1998; Wang et al., 2003; Byrnes and Griffin,1998). The production of DI particles in cultured cells can be con-trolled by using low MOI. Temperature does not seem to be anessential parameter in controlling viral infectivity in our in vitrosystem as titers obtained were similar whatever the incubationtemperature of infected cells (Table S3).

Genome analysis of CHIKV strains obtained after 30 cell passagesshowed that the alternately-passaged P30Alt strain and the seri-ally-passaged P30BHK21 strain on mammalian cells accumulated

Fig. 6. Dissemination of viral clones (A) and viral loads (B) in Ae. aegypti AAPT and Ae. albopictus ALPROV. Mosquitoes were orally infected with a blood-meal containing106.5 pfu/mL of each CHIKV clone differing by one substitution in E2 gene. To measure viral dissemination, viral titers were determined in two batches of ten mosquito headsat day 7 post-infection. ⁄⁄⁄p 6 0.001; ⁄⁄p 6 0.01; ⁄p 6 0.05; NS, non-significant after Chi square test (A) or Kruskal–Wallis test (B).

124 C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126

five amino-acid substitutions when examining the consensussequence. These substitutions were located in the nsP1, nsP3 and E2proteins(Table1). Theserially-passagedP30Aag2strainon mosquitocells accumulated just one amino-acid substitution in the E2glycoprotein (E2-N7S). Thus, mammalian and mosquito environ-ments do not represent equal partners in shaping CHIKV evolution.It has been demonstrated that mosquito cells tend to exert a minimaleffect on the evolution of some arboviruses such as dengue virus(Chen et al., 2003; Vasilakis et al., 2009). However, only analyzingthe consensus sequences tends to neglect minority variants that areimportant for generating CHIKV diversity during host alternation(Coffey and Vignuzzi, 2011). In our work, we compared the geneticdiversity at the E2 gene of ten biological clones isolated every fivepassages of each serial/alternate viral passages. Serially-passagedCHIKV produced more amino-acid substitutions compared to thealternately-passaged viruses (Table 2). Four amino-acid substitu-tions in the E2 glycoprotein were found at positions E2-7, E2-60,E2-64 and E2-208.

Interestingly, biological clones E2-64W/208E (BHK-21), E2-60D(Alternate) and E2-7N (Aag-2) showed a high dissemination rate in

Ae. aegypti, despite the parental strain (E1-226V) being poorlyassociated and disseminated by this mosquito species (Fig. 6A).In Ae. aegypti the P30Aag2 strain, which is different from the paren-tal strain by a single amino acid substitution (E2-N7S), and its de-rived viral clones (E2-7S and E2-7N), showed a dissemination ratealways higher than the parental strain (Figs. 5A and 6A). Concern-ing Ae. albopictus, infection with the P30Aag2 strain or its derivedclones provide contradictory results. Indeed, Ae. albopictus exhibitsa viral dissemination rate significantly lower when infected withthe P30Aag2 strain (Fig. 5B), or higher when infected with the viralclones E2-7S and E2-7N (Fig. 6B). The results shown in Fig. 2 sup-port these observations since the serial P30Aag2 strain (closer tothe parental strain than all other strains) was also the most singu-lar in terms of replication in all cell cultures used. We also showedthat two substitutions E2-W64R and E2-E208K detected very earlyfrom the 10th serial passage in BHK-21 cells (Table 2) tended tolower viral dissemination in Ae. aegypti without any effect in Ae.albopictus. In addition, the substitution E2-D60N detected fromthe 15th alternate passage tended to decrease viral disseminationin both mosquito species. This substitution has already been

C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126 125

suggested to result from the adaptation of CHIKV to replicate oncell cultures (Smit et al., 2002; Tsetsarkin et al., 2009).

In contrast to the E2-D60N substitution, the substitutionsE2-W64R and E2-E208K significantly lowered the disseminationof the E1-226V variant in Ae. aegypti. These two substitutions arelocated in close proximity to sites identified by (Tsetsarkin et al.,2009). Increased positive charge due to the replacement of anAsp residue in E2-64 and a Lys residue in E2-208 is likely to affectbinding with cell receptors (Strauss and Strauss, 1994). Indeed,passages of viruses in vertebrate cells can lead to select for bindingto heparan sulfates, mediated by a replacement by positive-chargesubstitutions in the E2 envelope glycoprotein (Brault et al., 2002;Klimstra et al., 1998). These amino-acid changes might affect mos-quito infectivity by disturbing the interactions between the virusand the receptors present on midgut cells (Myles et al., 2003). Itis tempting to suggest that the substitutions at the E2-64 andE2-208 tend to break the ability of the variant E1-226V to interactwith a cellular receptor on the midgut epithelial cells of Ae. aegyptiand not of Ae. albopictus. The position E2-208 is located in closeproximity to sites that previously have been shown to be involvedin adaptation of alphaviruses to a new host species (Brault et al.,2004; Anishchenko et al., 2006; Burness et al., 1988). Selectionby serial passages on Ae. aegypti-derived cells was not able to selectbasic residues in E2; this may explain the low replicative abilitiesof these viruses (Fig. 2).

Even if in our study we focus on the E2 amino-acid substitu-tions, we cannot exclude that minority variants or non-codingregions of the CHIKV genome may play a significant role in thedifferences that we have observed. For example, it has been dem-onstrated that the 30UTRs of alphaviruses play an important role ininteracting with cellular proteins to facilitate genome replicationin both mosquito and mammalian cells (Garneau et al., 2008;Sokoloski et al., 2010). In addition, a recent study suggests thatadaptation to mosquitoes is a major factor driving evolution ofthe CHIKV 30UTR (Chen et al., 2013). This certainly needs furtherinvestigation.

In conclusion, amino-acid changes in the E2 glycoprotein, W64Rand E208K, affect the capacity of the newly emerged variant ofCHIKV to disseminate in the mosquito Ae. aegypti and not in thevector Ae. albopictus. Interactions between the virus and a cellularreceptor on the mosquito midgut epithelial cells are likely to be af-fected. Our findings underscore the high capacity of RNA virusessuch as CHIKV to affect vector competence through minor geneticchanges. This contrasts with our previous study where phenotypicchanges were only found following large deletions in the viralgenome (Moutailler et al., 2011). These examples underline differ-ential strategies undergone by arboviruses to affect viral transmis-sion. Nevertheless, the substitutions W64R and E208K in the E2glycoprotein have not been identified in the current CHIKV strains,which could limit transmission of the E1-226V strain by Ae. aegyp-ti. Therefore, the E1-226V strain could continue to spread aroundthe world whatever the mosquito vector involved for transmission,Ae. aegypti or Ae. albopictus (Table S4).

Authors’ contributions

C.A.G. achieved experiments, analyzed data and drafted themanuscript. S.M. conducted viral passages. L.M. carried out miceexperiments. K.Z. helped in titration. V.C. and J.M.T. achievedsequencing experiments. F.R. and A.B.F. conceived the study andwrote the manuscript. All authors have read and approved themanuscript.

Acknowledgments

We wish to thank Anne-Sophie Delannoy-Vieillard and LaureDiancourt from the platform ‘‘Genotyping of pathogens and Public

Health’’ at the Institut Pasteur for technical support. We are in-debted to Thérèse Couderc for helping in experiments with mice,Philippe Desprès for providing viral strains and reagents, andYoann Madec for statistical analysis. We wish to thank Albin Fon-taine and Marie Vazeille for scientific and technical advices. Wealso wish to acknowledge Yannis Michalakis and Richard Paul fora critical reading of the manuscript. We are also thankful to PeterSahlins for corrections in English. We thank the ARS ‘‘AgenceRégionale de Santé’’ in the Indian Ocean and Didier Fontenille forproviding mosquito strains.

This study has received funding from the Institut Pasteur (ACIPGrant A-10-2009), the European Commission Framework ProgramSeven Award ‘‘InfraVec’’ (No. 228421), and the French Govern-ment’s Investissement d’Avenir program, Laboratoire d’Excellence‘‘Integrative Biology of Emerging Infectious Diseases’’ (Grant No.ANR-10-LABX-62-IBEID). C.A.G. was supported by the French Min-istry of Superior Education and Research and K.Z. by the EuropeanCommunity’s Seventh Framework Programme (FP7/2007–2013)under the project ‘‘VECTORIE’’, EC Grant Agreement No. 261466.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.meegid.2014.03.015.

References

Anishchenko, M., Bowen, R.A., Paessler, S., Austgen, L., Greene, I.P., Weaver, S.C.,2006. Venezuelan encephalitis emergence mediated by a phylogeneticallypredicted viral mutation. Proc. Natl. Acad. Sci. USA 103, 4994–4999.

Arias-Goeta, C., Mousson, L., Rougeon, F., Failloux, A.B., 2013. Dissemination andtransmission of the E1-226V variant of chikungunya virus in Aedes albopictusare controlled at the midgut barrier level. PLoS ONE 8, e57548.

Bagny, L., Delatte, H., Quilici, S., Fontenille, D., 2009. Progressive decrease in Aedesaegypti distribution in Reunion Island since the 1900s. J. Med. Entomol. 46,1541–1545.

Brackney, D.E., Scott, J.C., Sagawa, F., Woodward, J.E., Miller, N.A., Schilkey, F.D.,Mudge, J., Wilusz, J., Olson, K.E., Blair, C.D., Ebel, G.D., 2010. C6/36 Aedesalbopictus cells have a dysfunctional antiviral RNA interference response. PLoSNegl. Trop. Dis. 4, e856.

Brault, A.C., Powers, A.M., Holmes, E.C., Woelk, C.H., Weaver, S.C., 2002. Positivelycharged amino acid substitutions in the E2 envelope glycoprotein are associatedwith the emergence of Venezuelan equine encephalitis virus. J. Virol. 76, 1718–1730.

Brault, A.C., Powers, A.M., Ortiz, D., Estrada-Franco, J.G., Navarro-Lopez, R., Weaver,S.C., 2004. Venezuelan equine encephalitis emergence: enhanced vectorinfection from a single amino acid substitution in the envelope glycoprotein.Proc. Natl. Acad. Sci. USA 101, 11344–11349.

Burness, A.T., Pardoe, I., Faragher, S.G., Vrati, S., Dalgarno, L., 1988. Genetic stabilityof Ross River virus during epidemic spread in nonimmune humans. Virology167, 639–643.

Byrnes, A.P., Griffin, D.E., 1998. Binding of Sindbis virus to cell surface heparinsulfate. J. Virol. 72, 7349–7356.

Chen, W.J., Wu, H.R., Chiou, S.S., 2003. E/NS1 modifications of dengue 2 virus afterserial passages in mammalian and/or mosquito cells. Intervirology 46, 289–295.

Chen, R., Wang, E., Tsetsarkin, K.A., Weaver, S.C., 2013. Chikungunya virus 30

untranslated region: adaptation to mosquitoes and a population bottleneck asmajor evolutionary forces. PLoS Pathog. 9, e1003591.

Ciota, A.T., Kramer, L.D., 2010. Insights into arbovirus evolution and adaptation fromexperimental studies. Viruses 2, 2594–2617.

Ciota, A.T., Lovelace, A.O., Jia, Y., Davis, L.J., Young, D.S., Kramer, L.D., 2008.Characterization of mosquito-adapted West Nile virus. J. Gen. Virol. 89, 1633–1642.

Ciota, A.T., Jia, Y., Payne, A.F., Jerzak, G., Davis, L.J., Young, D.S., Ehrbar, D., Kramer,L.D., 2009. Experimental passage of St. Louis encephalitis virus in vivo inmosquitoes and chickens reveals evolutionarily significant virus characteristics.PLoS ONE 4, e7876.

Clarke, D.K., Duarte, E.A., Moya, A., Elena, S.F., Domingo, E., Holland, J., 1993. Geneticbottlenecks and population passages cause profound fitness differences in RNAviruses. J. Virol. 67, 222–228.

Cleaveland, S., Haydon, D.T., Taylor, L., 2007. Overviews of pathogen emergence:which pathogens emerge, when and why? Curr. Top. Microbiol. Immunol. 315,85–111.

Coffey, L.L., Vignuzzi, M., 2011. Host alternation of chikungunya virus increasesfitness while restricting population diversity and adaptability to novel selectivepressures. J. Virol. 85, 1025–1035.

126 C. Arias-Goeta et al. / Infection, Genetics and Evolution 24 (2014) 116–126

Coffey, L.L., Vasilakis, N., Brault, A.C., Powers, A.M., Tripet, F., Weaver, S.C., 2008.Arbovirus evolution in vivo is constrained by host alternation. Proc. Natl. Acad.Sci. USA 10, 6970–6975.

Coffey, L.L., Beeharry, Y., Borderia, A.V., Blanc, H., Vignuzzi, M., 2011. Arbovirus highfidelity variant loses fitness in mosquitoes and mice. Proc. Natl. Acad. Sci. USA10, 16038–16043.

Cooper, L.A., Scott, T.W., 2001. Differential evolution of eastern equine encephalitisvirus populations in response to host cell type. Genetics 157, 1403–1412.

Couderc, T., Chrétien, F., Schilte, C., Disson, O., Brigitte, M., Guivel-Benhassine, F.,Touret, Y., Barau, G., Cayet, N., Schuffenecker, I., Desprès, P., Arenzana-Seisdedos, F., Michault, A., Albert, M.L., Lecuit, M., 2008. A mouse model forChikungunya: young age and inefficient type-I interferon signaling are riskfactors for severe disease. PLoS Pathog. 4, e29.

de Lamballerie, X., Leroy, E., Charrel, R.N., Tsetsarkin, K., Higgs, S., Gould, E.A., 2008.Chikungunya virus adapts to tiger mosquito via evolutionary convergence: asign of things to come? Virol. J. 5, 33.

Deardorff, E.R., Fitzpatrick, K.A., Jerzak, G.V., Shi, P.Y., Kramer, L.D., Ebel, G.D., 2011.West Nile virus experimental evolution in vivo and the trade-off hypothesis.PLoS Pathog. 7, e1002335.

Desmyter, J., Melnick, J.L., Rawls, W.E., 1968. Defectiveness of interferon productionand of rubella virus interference in a line of African green monkey kidney cells(Vero). J. Virol. 2, 955–961.

Domingo, E., Holland, J.J., 1997. RNA virus mutations and fitness for survival. Annu.Rev. Microbiol. 51, 151–178.

Domingo, E., Sheldon, J., Perales, C., 2012. Viral quasispecies evolution. Microbiol.Mol. Biol. Rev. 76, 159–216.

Drake, J.W., Holland, J.J., 1999. Mutation rates among RNA viruses. Proc. Natl. Acad.Sci. USA 96, 13910–13913.

Eigen, M., 1996. On the nature of virus quasispecies. Trends Microbiol. 4, 216–218.Elena, S.F., Bedhomme, S., Carrasco, P., Cuevas, J.M., de la Iglesia, F., Lafforgue, G.,

Lalic, J., Pròsper, A., Tromas, N., Zwart, M.P., 2011. The evolutionary genetics ofemerging plant RNA viruses. Mol. Plant Microbe Interact. 24, 287–293.

Fallon, A.M., Sun, D., 2001. Exploration of mosquito immunity using cells in culture.Insect Biochem. Mol. Biol. 31, 263–278.

Fitzpatrick, K.A., Deardorff, E.R., Pesko, K., Brackney, D.E., Zhang, B., Bedrick, E., Shi,P.Y., Ebel, G.D., 2010. Population variation of West Nile virus confers a host-specific fitness benefit in mosquitoes. Virology 404, 89–95.

Garneau, N.L., Sokoloski, K.J., Opyrchal, M., Neff, C.P., Wilusz, C.J., Wilusz, J., 2008.The 30 untranslated region of sindbis virus represses deadenylation of viraltranscripts in mosquito and Mammalian cells. J. Virol. 82, 880–892.

Greene, I.P., Wang, E., Deardorff, E.R., Milleron, R., Domingo, E., Weaver, S.C., 2005.Effect of alternating passage on adaptation of Sindbis virus to vertebrate andinvertebrate cells. J. Virol. 79, 14253–14260.

Holland, J., Spindler, K., Horodyski, F., Grabau, E., Nichol, S., VandePol, S., 1982. Rapidevolution of RNA genomes. Science 215, 1577–1585.

Holm, S., 1979. A simple sequentially rejective multiple test procedure. Scand. J.Stat. 6, 65–70.

Igarashi, A., 1978. Isolation of a Singh’s Aedes albopictus cell clone sensitive todengue and chikungunya viruses. J. Gen. Virol. 40, 531–544.

Jerzak, G., Bernard, K.A., Kramer, L.D., Ebel, G.D., 2005. Genetic variation in West Nilevirus from naturally infected mosquitoes and birds suggests quasispeciesstructure and strong purifying selection. J. Gen. Virol. 86, 2175–2183.

Klimstra, W.B., Ryman, K.D., Johnston, R.E., 1998. Adaptation of Sindbis virus to BHKcells selects for use of heparan sulfate as an attachment receptor. J. Virol. 72,7357–7366.

Lauring, A.S., Andino, R., 2010. Quasispecies theory and the behavior of RNA viruses.PLoS Pathog. 6, e1001005.

Lin, S.R., Hsieh, S.C., Yueh, Y.Y., Lin, T.H., Chao, D.Y., Chen, W.J., King, C.C., Wang,W.K., 2004. Study of sequence variation of dengue type 3 virus in naturallyinfected mosquitoes and human hosts: implications for transmission andevolution. J. Virol. 78, 12717–12721.

MacPherson, I., Stoker, M., 1962. Polyoma transformation of hamster cell clones –an investigation of genetic factors affecting cell competence. Virology 16, 147–151.

Martin, E., Moutailler, S., Madec, Y., Failloux, A.B., 2010. Differential responses of themosquito Aedes albopictus from the Indian Ocean region to two chikungunyaisolates. BMC Ecol. 10, 8.

Moncayo, A.C., Fernandez, Z., Ortiz, D., Diallo, M., Sall, A., Hartman, S., Davis, C.T.,Coffey, L., Mathiot, C.C., Tesh, R.B., Weaver, S.C., 2004. Dengue emergence andadaptation to peridomestic mosquitoes. Emerg. Infect. Dis. 10, 1790–1796.

Moudy, R.M., Meola, M.A., Morin, L.L., Ebel, G.D., Kramer, L.D., 2007. A newlyemergent genotype of West Nile virus is transmitted earlier and moreefficiently by Culex mosquitoes. Am. J. Trop. Med. Hyg. 77, 365–370.

Moutailler, S., Roche, B., Thiberge, J.M., Caro, V., Rougeon, F., Failloux, A.B., 2011.Host alternation is necessary to maintain the genome stability of Rift valleyfever virus. PLoS Negl. Trop. Dis. 5, e1156.

Myles, K.M., Pierro, D.J., Olson, K.E., 2003. Deletions in the putative cell receptorbinding domain of sindbis virus strain MRE16 E2 glycoprotein reduce midgutinfectivity in Aedes aegypti. J. Virol. 77, 8872–8881.

Novella, I.S., Hershey, C.L., Escarmis, C., Domingo, E., Holland, J.J., 1999. Lack ofevolutionary stasis during alternating replication of an arbovirus in insect andmammalian cells. J. Mol. Biol. 287, 459–465.

Novella, I.S., Presloid, J.B., Smith, S.D., Wilke, C.O., 2011. Specific and nonspecifichost adaptation during arboviral experimental evolution. J. Mol. Microbiol.Biotechnol. 21, 71–81.

Otsuki, K., Maeda, J., Yamamoto, H., Tsubokura, M., 1979. Studies on avian infectiousbronchitis virus (IBV). III. Interferon induction by and sensitivity to interferon ofIBV. Arch. Virol. 60, 249–255.

Peleg, J., 1968. Growth of arboviruses in primary tissue culture of Aedes aegyptiembryos. Am. J. Trop. Med. Hyg. 17, 219–223.

Pfeiffer, J.K., Kirkegaard, K., 2005. Increased fidelity reduces poliovirus fitness andvirulence under selective pressure in mice. PLoS Pathog. 1, e11.

Powers, A.M., Logue, C.H., 2007. Changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus. J. Gen. Virol. 88, 2363–2377.

Quinones-Mateu, M.E., Arts, E.J., 2006. Virus fitness: concept, quantification, andapplication to HIV population dynamics. Curr. Top. Microbiol. Immunol. 299,83–140.

Smit, J.M., Waarts, B.L., Kimata, K., Klimstra, W.B., Bittman, R., Wilschut, J., 2002.Adaptation of alphaviruses to heparan sulfate: interaction of Sindbis andSemliki forest viruses with liposomes containing lipid-conjugated heparin. J.Virol. 76, 10128–10137.

Sokoloski, K.J., Dickson, A.M., Chaskey, E.L., Garneau, N.L., Wilusz, C.J., Wilusz, J.,2010. Sindbis virus usurps the cellular HuR protein to stabilize its transcriptsand promote productive infections in mammalian and mosquito cells. Cell HostMicrobe 8, 196–207.

Steinhauer, D.A., Holland, J.J., 1987. Rapid evolution of RNA viruses. Annu. Rev.Microbiol. 41, 409–433.

Steinhauer, D.A., Domingo, E., Holland, J.J., 1992. Lack of evidence for proofreadingmechanisms associated with an RNA virus polymerase. Gene 122, 281–288.

Strauss, J.H., Strauss, E.G., 1994. The alphaviruses: gene expression, replication, andevolution. Microbiol. Rev. 58, 491–562.

Tsetsarkin, K.A., Weaver, S.C., 2011. Sequential adaptive mutations enhanceefficient vector switching by Chikungunya virus and its epidemic emergence.PLoS Pathog. 7, e1002412.

Tsetsarkin, K.A., Vanlandingham, D.L., McGee, C.E., Higgs, S., 2007. A single mutationin chikungunya virus affects vector specificity and epidemic potential. PLoSPathog. 3, e201.

Tsetsarkin, K.A., McGee, C.E., Volk, S.M., Vanlandingham, D.L., Weaver, S.C., Higgs, S.,2009. Epistatic roles of E2 glycoprotein mutations in adaption of chikungunyavirus to Aedes albopictus and Ae. aegypti mosquitoes. PLoS ONE 4, e6835.

Vasilakis, N., Deardorff, E.R., Kenney, J.L., Rossi, S.L., Hanley, K.A., Weaver, S.C., 2009.Mosquitoes put the brake on arbovirus evolution: experimental evolutionreveals slower mutation accumulation in mosquito than vertebrate cells. PLoSPathog. 5, e1000467.

Vazeille, M., Moutailler, S., Coudrier, D., Rousseaux, C., Khun, H., Huerre, M., Thiria, J.,Dehecq, J.S., Fontenille, D., Schuffenecker, I., Despres, P., Failloux, A.B., 2007. TwoChikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibitdifferent patterns of infection in the mosquito, Aedes albopictus. PLoS ONE 2,e1168.

Wang, E., Ni, H., Xu, R., Barrett, A.D., Watowich, S.J., Gubler, D.J., Weaver, S.C., 2000.Evolutionary relationships of endemic/epidemic and sylvatic dengue viruses. J.Virol. 74, 3227–3234.

Wang, E., Brault, A.C., Powers, A.M., Kang, W., Weaver, S.C., 2003.Glycosaminoglycan binding properties of natural Venezuelan equineencephalitis virus isolates. J. Virol. 77, 1204–1210.

Weaver, S.C., Brault, A.C., Kang, W., Holland, J.J., 1999. Genetic and fitness changesaccompanying adaptation of an arbovirus to vertebrate and invertebrate cells. J.Virol. 73, 4316–4326.

Yasumura, Y., Kawakita, M., 1963. The research for the SV40 by means of tissueculture technique. Nippon Rinsho 21, 1201–1219.