JOURNAL OF VIROLOGY, July 2010, p. 6497–6504 Vol. 84, No.

13
0022-538X/10/$12.00 doi:10.1128/JVI.01603-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Genome-Scale Phylogenetic Analyses of Chikungunya Virus

Reveal Independent Emergences of Recent Epidemics
and Various Evolutionary Rates䌤‡

Downloaded from http://jvi.asm.org/ on August 12, 2019 at ICDDR B CENTER FOR HLTH &
Sara M. Volk,1†§ Rubing Chen,1† Konstantin A. Tsetsarkin,1 A. Paige Adams,1 Tzintzuni I. Garcia,2¶
Amadou A. Sall,3 Farooq Nasar,1 Amy J. Schuh,1 Edward C. Holmes,4 Stephen Higgs,1
Payal D. Maharaj,5储 Aaron C. Brault,5储 and Scott C. Weaver1*
Center for Biodefense and Emerging Infectious Diseases and Department of Pathology,1 and Department of Biochemistry and
Molecular Biology,2 University of Texas Medical Branch, Galveston, Texas; Institut Pasteur de Dakar, Dakar, Senegal3; Center for
Infectious Disease Dynamics, Department of Biology, Pennsylvania State University, University Park, Pennsylvania4; and Center for
Vector-Borne Diseases and Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine,
University of California, Davis, California5
Received 2 August 2009/Accepted 29 March 2010

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia,
causing human febrile illness accompanied by severe, chronic joint pain. In Africa, epidemic emergence of
CHIKV involves the transition from an enzootic, sylvatic cycle involving arboreal mosquito vectors and
nonhuman primates, into an urban cycle where peridomestic mosquitoes transmit among humans. In Asia,
however, CHIKV appears to circulate only in the endemic, urban cycle. Recently, CHIKV emerged into the
Indian Ocean and the Indian subcontinent to cause major epidemics. To examine patterns of CHIKV evolution
and the origins of these outbreaks, as well as to examine whether evolutionary rates that vary between enzootic
and epidemic transmission, we sequenced the genomes of 40 CHIKV strains and performed a phylogenetic
analysis representing the most comprehensive study of its kind to date. We inferred that extant CHIKV strains
evolved from an ancestor that existed within the last 500 years and that some geographic overlap exists between
two main enzootic lineages previously thought to be geographically separated within Africa. We estimated that
CHIKV was introduced from Africa into Asia 70 to 90 years ago. The recent Indian Ocean and Indian
subcontinent epidemics appear to have emerged independently from the mainland of East Africa. This finding
underscores the importance of surveillance to rapidly detect and control African outbreaks before exportation
can occur. Significantly higher rates of nucleotide substitution appear to occur during urban than during
enzootic transmission. These results suggest fundamental differences in transmission modes and/or dynamics
in these two transmission cycles.

Chikungunya virus (CHIKV; Togaviridae: Alphavirus) is an with those of dengue and because CHIKV is transmitted sym-
arbovirus (arthropod-borne virus) vectored by Aedes mosqui- patrically in urban areas by the same mosquito vectors, it is
toes to humans in tropical and subtropical regions of Africa grossly underreported in the absence of laboratory diagnostics
and Asia (Fig. 1; reviewed in references 26 and 46). CHIKV (10, 37).
has a single-stranded, positive-sense RNA genome of ⬃12 kb CHIKV was first isolated during a 1953 outbreak in present-
and causes chikungunya fever (CHIK), a febrile illness associ- day Tanzania by Ross (48, 49). Since then, outbreaks have
ated with severe arthralgia and rash (2, 15, 31, 35); the name is been documented in Africa and Asia, including the Indian
derived from a Bantu language word describing the severe subcontinent (Fig. 1) (1, 4). In 2005, CHIKV emerged from
arthritic signs (32), which can persist for years. Thus, CHIK has East Africa to cause an explosive urban epidemic in popular
enormous economic costs in addition to its public health im- tourist island destinations in the Indian Ocean (Fig. 1; re-
pact (9). Because the signs and symptoms of CHIK overlap viewed in reference 31). In late 2005, CHIKV spread into the
Indian subcontinent, where millions of people have been af-
fected (5). However, the geographic source of spread into
* Corresponding author. Mailing address: University of Texas Med-
India, from the mainland of Africa or from the Indian Ocean
ical Branch, 301 University Blvd., Galveston, TX 77555-0609. Phone:
(409) 747-0758. Fax: (409) 747-2429. E-mail: [email protected]. Islands, has not been delineated. India had seen large epidem-
† S.M.V. and R.C. contributed equally to this study. ics of CHIK in the past (reviewed in reference 30), but CHIKV
‡ Supplemental material for this article may be found at http://jvi apparently disappeared during the 1970s (5). Since 2006,
.asm.org/.
CHIKV has been imported into Europe and the western hemi-
§ Present address: Department of Biology, Our Lady of the Lake
University, San Antonio, TX 78207. sphere (including the United States) via many viremic travel-
¶ Present address: Xiphophorus Genetic Stock Center, Department ers, and an epidemic was initiated in Italy by a traveler from
of Chemistry and Biochemistry, Texas State University, San Marcos, India (4, 11, 47). The dramatic spread since 1980 of dengue
TX 78666.
viruses (DENV) throughout tropical America, via the same
储 Present address: Division of Vector-Borne Diseases, Centers for
Disease Control and Prevention, Fort Collins, CO 80521. vectors, portends the severity of the public health problem if
䌤
Published ahead of print on 21 April 2010. CHIKV becomes established in the western hemisphere.

6497
6498 VOLK ET AL. J. VIROL.

Downloaded from http://jvi.asm.org/ on August 12, 2019 at ICDDR B CENTER FOR HLTH &
Country with endemic/epidemic case Seychelles
Country with inported case Anbian Island, Indonesia
Comoros
West African Lineage Mayotte
ECSA Lineage Mauritius
Epidemic Lineage La Reunion
Asian Lineage

FIG. 1. Distribution of the CHIKV strains used in this study. The map, based on a world map template from http://www.presentationmagazine
.com, was edited with permission.

The first phylogenetic analysis of CHIKV (45) identified forth called enzootic) and urban/endemic/epidemic transmis-
three geographically associated genotypes: the West African sion cycles (henceforth called epidemic) such as seasonality of
(WAf), East/Central/South African (ECSA), and Asian geno- vector larval habitats, vertebrate host abundance and herd
types. More recent analyses indicate that the recent Indian immunity, and vector host preferences, prompted us to hypoth-
Ocean and Indian strains form a monophyletic group within esize that the evolutionary dynamics of CHIKV may differ
the ECSA lineage (5, 12, 14, 27, 40, 51, 52). However, most between the two transmission cycles. To test this hypothesis, to
CHIKV phylogenetic studies (1, 14, 28, 29, 38, 40, 41, 47, 52) provide more robust estimates of the evolutionary relation-
have utilized only partial sequences from the envelope glyco- ships among the CHIKV strains including the sources of the
protein E1 gene, preventing a robust assessment of some of the recent epidemics, and to elucidate the temporal and spatial
relationships among strains and of their evolutionary dynam- history of CHIKV evolution, we performed an extensive, ge-
ics. nome-scale phylogenetic analysis, utilizing complete open
The CHIKV strains represented in different geographic lin- reading frame (ORF) sequences of a large collection of 80
eages apparently circulate in different ecological cycles. In isolates with broad temporal, spatial, and host coverage.
Asia, CHIKV appears to circulate primarily in an urban trans-
mission cycle involving the peridomestic mosquitoes Aedes ae- MATERIALS AND METHODS
gypti and A. albopictus, as well as humans (25, 45). Asian Virus samples. CHIKV strains listed in Table S1 in the supplemental material
epidemics typically infect thousands-to-millions of people over were either obtained from the World Reference Center for Emerging Viruses
the course of several years (46). In contrast, African CHIKV and Arboviruses at the University of Texas Medical Branch, Galveston, TX, or
circulates primarily in a sylvatic/enzootic cycle, transmitted by from the Institut Pasteur de Dakar, Senegal. Viruses were passaged in C6/36 A.
albopictus cells, concentrated with polyethylene glycol (7% [wt/vol]) and NaCl
arboreal primatophilic Aedes mosquitoes (e.g., A. furcifer and (2.3% [wt/vol]), and RNA was extracted by using either TRIzol (Invitrogen,
A. africanus) and probably relies on nonhuman primates as Carlsbad, CA) or the QIAamp viral RNA minikit (Qiagen, Valencia, CA) ac-
reservoir hosts (reviewed in reference 16). Epidemics in rural cording to the manufacturers’ protocols.
Africa usually occur on a much smaller scale than in Asia, RT-PCR and sequencing. Eight or nine overlapping PCR amplicons were
generated from viral RNA using the Titan One Tube reverse transcription-PCR
likely a result of the lower human population densities, and
system (Roche, Mannheim, Germany) according to the manufacturer’s protocol.
possibly more stable herd immunity. Although the assignments The amplicons were subsequently gel purified and sequenced by using a BigDye
of “urban” and “sylvatic/enzootic” are based on the most com- Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA).
mon mode of transmission, CHIKV strains of African origin Sequencing was performed in an ABI Prism model 3100 genetic analyzer (Ap-
are capable of urban transmission by A. aegypti and A. albop- plied Biosystems, Foster City, CA), and the sequences were edited and assem-
bled in Sequencher v4.9 (Gene Codes, Ann Arbor, MI), and deposited in the
ictus, as evidenced by outbreaks in the Democratic Republic of GenBank database (see Table S1 in the supplemental material). Primer se-
the Congo (41), Nigeria (36), Kenya (27), and Gabon (42). The quences and specific PCR and sequencing protocols are available from the
ecological differences between the sylvatic/enzootic (hence- authors.
VOL. 84, 2010 CHIKV EVOLUTION IN TWO TRANSMISSION CYCLES 6499

Phylogenetic analyses. Newly generated CHIKV genomic sequences, as well synonymous (dS) nucleotide substitutions per site (dN/dS ratio) for each clade
as those available from the GenBank library (excluding vaccine and cloning using the “one-ratio” model from the CODEML program available within the
vector strains), along with that of o’nyong-nyong virus (ONNV; strains Gulu, PAML package (63), using the subtrees of the total ML tree indicated above. In
Igbo Ora, and SG650, used as an outgroup) were aligned by using MUSCLE (20) addition, the selection pressures on external and internal branches of each ML
and manually adjusted in Se-Al (available at http://tree.bio.ed.ac.uk/software phylogeny were estimated separately using the “two-ratio” model available in
/seal/) according to amino acid sequence alignments to preserve codon homol- CODEML. Similarly, the numbers and locations of positively and negatively
ogy. Due to the ambiguous alignments in the 3⬘ untranslated region (3⬘UTRs) selected sites were estimated by using the internal fixed effects likelihood method
and rapid evolution in the other UTR, only open reading frames (ORFs) were available in the HYPHY package (42a).
adopted in further analysis. This led to an alignment of 11,319 nucleotides,

Downloaded from http://jvi.asm.org/ on August 12, 2019 at ICDDR B CENTER FOR HLTH &
containing 80 CHIKV strains and 3 ONNV isolates. To search for any potential
recombination event in CHIKV that could affect the phylogenetic structure,
RESULTS
genomic sequences were screened exhaustively and triplet-by-triplet in RDP
version 3.41 (34) and using a suite of different recombination detection methods Genetic diversity of CHIKV. Forty CHIKV genomes were
using RDP (33), Chimaera (43), MaxChi (57), 3Seq (8), and GENECONV (39).
Recombination with significantly positive hits was not found in our data set,
sequenced and aligned with those available in GenBank. Ex-
including in the high passage isolates. cluding the 5⬘ and 3⬘ 20 nucleotides (nt) that were not se-
Phylogenetic trees were inferred by using both the maximum-likelihood (ML) quenced, the genome length varied among and within geo-
method in the PAUP* v4.0b package (58) and the Metropolis-coupled Markov graphic lineages, with those in the ECSA lineage being shorter
Chain Monte Carlo (MCMCMC) method in MrBayes v3.1.2 (24). MODELTEST (11,557 to 11,789 nt) than the WAf (11,843 to 11,881 nt) and
(44) was used to select the best-fit model for the ML analyses. MrBayes analyses
used the GTR⫹I⫹⌫4 model; in addition, the nucleotide data were partitioned by
Asian (11,777 to 11,999 nt) strains. Nucleotide differences were
three codon positions, and substitution parameters were allowed to vary across found in all genes, and the most variable genome regions
partitions. The analysis used three hot chains and one cold chain and ran for 10 included the 5⬘ and 3⬘ UTRs, as well as the 26S junction region.
million generations with 25% burnin; the “sump” command and Tracer v1.4.1 The ORFs were highly conserved, with occasional indels ob-
(http://tree.bio.ed.ac.uk/software/tracer/) were used to ensure samples were taken
served in high-passage strains. The highly divergent UTRs
after likelihoods had stabilized.
To examine the possible advantage of using complete genome sequences in the made accurate alignments impossible, and the UTRs were
phylogenetic study, we also inferred an ML tree based on the E1 gene and therefore excluded from the phylogenetic analyses. We found
compared it to the ML phylogeny of complete ORF. To determine whether these poly(A) insertions in the 3⬘UTRs of two ECSA strains (Fig. 2)
trees differ significantly in topology, we used the Shimodaira-Hasegawa (SH) test in addition to the Ross strain (5). The insertion in the Senegal
implemented in PAUP.
Rates of nucleotide substitution and ttMRCA. We used the Bayesian Markov
bat strain, which is located in the ECSA group, is in a different
Chain Monte Carlo (MCMC) method available in BEAST v1.5.3 (18) to estimate location from the other two, suggesting their independent gen-
evolutionary rates and times to the most recent common ancestors (tMRCA) for eration.
CHIKV overall and for each of the individual clades, namely, WAf, ECSA, Origin and divergence of geographically related clades. Sim-
Asian, and the recent epidemic group. To avoid artifacts due to laboratory
ilar to previous findings (47), our phylogenetic trees (shown in
adaptation, high-passage strains (Ross and S27) were excluded from this analysis,
as well as those without clear sample year information (see Table S1 in the Fig. 2 and see Fig. S1 and S2 in the supplemental material) all
supplemental material). The strains Angola/M2022/1962, India/MH4/2000, and included three distinct CHIKV clades, namely, ECSA, West
India/ALSA-1/1986 were also omitted because of the potential that they were the Africa, and Asian, with the recent Indian Ocean basin out-
result of contamination or high passage, as suggested by either a suspicious break forming a monophyletic lineage descendant from the
terminal branch length or phylogenetic position (see Fig. S1 and S2 in the
supplemental material). However, strains with low passage histories (⬍10) were
ECSA clade. The divergence of each distinct lineage reflected,
used in analysis because previous studies with alphaviruses have shown that these to some extent, the path of global transmission and occasional
small numbers of passages introduce few mutations (13, 62) and should therefore outbreaks. According to our estimate, the currently circulating
have little or no effect on coalescent studies. All strains were dated according to CHIKV strains have an ancestor that existed within the last
the year and month (if known) of their collection; strains with only a known
500 years, with 95% HPD values of the most recent common
collection year were assigned the median-year value for July. Due to the short
time scale of the recent Indian Ocean basin epidemic group, only sequences with ancestor (MRCA) ranging from 169 to 436 years ago. Inter-
a known sample month were included in evolutionary rate and tMRCA estima- estingly, despite their close geographic distance, the two Afri-
tions. This led to a total data set of 80 genomic CHIKV sequences, including 21 can lineages did not cluster together, indicating limited genetic
Asian, 11 WAf, 9 ECSA, and 39 recent epidemic strains. BEAST analysis was exchange between the two lineages in Africa. The only excep-
performed for the combined data set and for individual clades, based on a
tion was a 1963 bat isolate from Senegal, which grouped in the
relaxed molecular clock (uncorrelated lognormal) and the SRD06 nucleotide
substitution model that has been suggested to be superior to other models when ECSA clade. This finding is the first to suggest that the main
used with ORFs in RNA viral genomes (54). All analyses also used a Bayesian West African and ECSA lineages may overlap spatially in the
skyline coalescent tree prior (19), which imposes the fewest demographic as- enzootic cycle, at least on occasion.
sumptions, and because estimating demographic history was not an aim of the The divergence of the ECSA and Asian lineages occurred
present study. In each case, MCMC chains were run for a sufficient time to
achieve convergence (accessed using the Tracer program; http://tree.bio.ed.ac
within the last 150 years (95% HPD: 1879 to 1927 AD), with
.uk/software/tracer). Statistical uncertainty in parameter estimates is reflected as the Asia group splitting into two clades: an Indian lineage,
the 95% highest probability density (HPD) values. The maximum clade credi- which likely went extinct (5, 30), and a Southeast Asian lin-
bility tree across all of the plausible trees generated by BEAST was then com- eage, which is probably still circulating. The Southeast Asian
puted by using the TreeAnnotator program available in BEAST package, with
lineage suggests a remarkable spatial and temporal pattern,
the first 10% of trees removed as burn-in. To assess the reliability of our
substitution rate and tMRCA estimates and to determine the extent of the spreading from Thailand to Indonesia, and then to the Philip-
temporal structure of the sequence data, we also performed a regression analysis pines, and more recently to Malaysia (Fig. 2). This temporal
of tree root-to-tip genetic distance against sampling dates using the program branching pattern was similar to patterns observed in some
Path-O-Gen (http://tree.bio.ed.ac.uk/software/pathogen/) (17) for each lineage DENV phylogenies (23) and in other alphaviruses such as
based on the corresponding phylogeny excised from the ML tree of 80 CHIKV
strains.
eastern equine encephalitis virus in North America (6).
Selection pressures. To investigate the nature of selection pressures that may Similarly, the recent Indian Ocean basin outbreak that be-
act on CHIKV, we estimated the average numbers of nonsynonymous (dN) and gan in 2004 apparently originated from the ECSA group as
6500 VOLK ET AL. J. VIROL.

2003/01 [2001/12,2003/12]
Indian Ocean
1 Outbreak

Downloaded from http://jvi.asm.org/ on August 12, 2019 at ICDDR B CENTER FOR HLTH &
1964 [1958,1971]
1
C

1935 [1924,1943]
1 SA_SAH2123_1976
ECSA
SA_AR18211_1976 (Ae. furcifer)
0.9878 Bangladesh_0810aTw_2008 **
Mombasa * SA_Vereeniging_1956
5

1904 [1879,1927]
1

0.9999 Lamu Asia

Comoros_Mar-J_2005

1713 [1573,1840] 1953 [1948,1956]

1 1

1956 [1952,1960] West Africa

1

1750 1800 1850 1900 1950 2000 (year)

FIG. 2. Maximum clade credibility (MCC) tree of 80 CHIKV strains. The four major lineages are highlighted with different branch colors, with
the sample origin highlighted by the corresponding color. The estimated 95% HPD values for most recent common ancestors are labeled beside
the node and are also indicated by the thick blue horizontal node bars. The numbers adjacent to nodes indicate Bayesian posterior probability
values. For clarity, the 2004 to 2009 epidemic clade is shown enlarged in the inset at upper left. Sequences with internal poly(A) insertion in the
3⬘UTR are marked with an asterisk (*). Strains are labeled as follows: location_strain name_date (year) of collection.

shown in Fig. 2, with the MRCA dating back to about 2002 Interestingly, a mosquito isolate, MH4 from 2000, which was
(95% HPD: December 2001 to December 2003). Two distinct collected in India, fell within the ECSA clade (see Fig. S1 and
lineages were identified for the Indian Ocean and Indian sub- S2 in the supplemental material), suggesting a CHIKV intro-
continent strains (as well as strains exported from these re- duction from Africa into India at least 5 years before the 2005
gions). The Indian subcontinent clade was rooted by a main- epidemic. However, the high (99.4%) nucleotide identity be-
land Kenya 2004 strain, while the Indian Ocean clade also tween Uganda/Ag4155/1982 and India/MH4/2000 was incon-
contains a 2004 Kenyan strain (Lamu Island), indicating inde- sistent with their nearly 20-year difference in collection dates,
pendent emergences from East Africa into the Indian Ocean given our estimates of CHIKV nucleotide substitution rates
and the Indian subcontinent. (Table 1). Given the overall strong temporal pattern shown in
The ECSA clade has been sampled in a broad geographic the phylogenetic trees, we suspect that India/MH4/2000 might
range since the first identification of CHIKV in 1953, with the be the result of contamination.
MRCA of available isolates dating to from 1924 to 1943 (95% Evolutionary patterns among CHIKV lineages. The evolu-
HPDs). In contrast to the clear pattern of geographic spread tionary rates estimated by using the Bayesian MCMC method
exhibited by the Asian lineage and the recent Indian Ocean for the complete CHIKV data set and individual lineages are
epidemic clades, there was no clear geographic pattern of summarized in Table 1. The overall nucleotide substitution
ECSA lineage spread. Due to the limited sample size, it is not rate was estimated as 4.33 ⫻ 10⫺4 nucleotide substitutions per
clear in what mode and to what extent this lineage has been site per year (subs/nt/year). However, the rates estimated for
circulating in eastern, central, and southern Africa. each lineage exhibited considerable variation, with those for
VOL. 84, 2010 CHIKV EVOLUTION IN TWO TRANSMISSION CYCLES 6501

TABLE 1. Rates of nucleotide substitution of chikungunya virus lected codons if unique, adaptive mutations occur, three pos-
Parameter CHIK80 Asian21 ECSA9 WAf11 Epidemic39 itively selected codons were also observed in the epidemic
lineage, comprising two codons in the capsid protein gene
Mean rate 4.33E-04 4.16E-04 2.30E-04 2.39E-04 8.41E-04
95% HPD lower 3.15E-04 3.26E-04 1.37E-04 1.98E-04 5.78E-04 (codons 23 and 27) and one in the E1 envelope glycoprotein
95% HPD upper 5.62E-04 5.02E-04 3.24E-04 2.84E-04 1.09E-03 gene (codon 226); the latter plays a crucial role in CHIKV
Coefficient of 0.44 0.63 0.11 0.08 0.44
variation
adaptation to A. albopictus (59, 61). However, these adaptive
mutations can only partially explain the elevated dN/dS value
in this lineage, since only 16 negatively selected codons were

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observed in the epidemic lineage, whereas many more (i.e., 64
the epidemic lineages significantly higher than those estimated to 96) were observed in other groups. Therefore, the elevated
for the enzootic lineages. In particular, the Asian lineage ex- evolutionary rate in the Indian Ocean epidemic lineage is most
hibited a significantly higher substitution rate (i.e., nonover- likely due to the presence of transient deleterious mutations
lapping HPD values) (4.16 ⫻ 10⫺4 subs/nt/yr; 95% HPD: 3.26 not seen in the other lineages. Finally, it was noteworthy that
to 5.02 ⫻ 10⫺4 subs/nt/year) than the WAf (2.39 ⫻ 10⫺4 subs/ dN/dS was also elevated in the Asian group compared to the
nt/year; 95% HPD: 1.98 to 2.84 ⫻ 10⫺4 subs/nt/year) and ECSA and WAf lineages (Table 2). This may indicate different
ECSA lineages (2.30 ⫻ 10⫺4 subs/nt/year; 95% HPD: 1.37 to selection pressures acting on the epidemic versus enzootic
3.24 ⫻ 10⫺4 subs/nt/year). The Indian Ocean epidemic lineage transmission cycles.
yielded an even higher rate estimate (8.46 ⫻ 10⫺4 subs/nt/year; Comparison of complete genomic and partial E1 sequences
95% HPD: 5.81 ⫻ 10⫺4 to 1.09 ⫻ 10⫺3 subs/nt/year). This in phylogenetic reconstructions. Importantly, the ML tree
widespread rate variation was confirmed by using root-to-tip based on E1 gene sequences revealed a topology different from
linear regression, with all lineages showing strong clocklike that based on the complete ORFs, especially for the ECSA
behavior (as reflected in the correlation coefficient values; see lineage, which did not form a monophyletic group in the
Fig. S3 in the supplemental material). former data set (see Fig. S3 in the supplemental material). A
It is possible, however, that the intensive sampling of se- significant difference between the E gene and complete ORF
quences within a short time span from the recent epidemic may tree topologies was also apparent in the SH test (P ⬍ 0.001).
have included many transient deleterious mutations (i.e., se- Since the complete ORFs tree is clearly more accurate, pos-
quence polymorphisms) that would later be purged by purify- sessing more variable and phylogenetic informative sites, this
ing selection and therefore not persist over longer time peri- analysis shows that the E gene alone is inadequate to fully
ods. This, in turn, would artificially increase the substitution resolve the phylogenetic history of CHIKV.
rate for the Indian Ocean lineage. To test this hypothesis, we
measured selection pressures among the four major CHIKV
DISCUSSION
lineages. This analysis revealed a significantly higher overall
dN/dS value in the Indian Ocean epidemic lineage (0.285) CHIKV origins. CHIKV has likely been circulating in Africa
compared to the others (0.066 to 0.125), which is compatible and Asia for hundreds of years or even longer. A suspected
with the presence of transient, mildly deleterious mutations in CHIK epidemic was reported in 1779 (10), and focal epidemics
the former group. A similar hypothesis was proposed to ex- have been documented occasionally throughout the second
plain the higher dN/dS values for swine-origin influenza A half of the 20th century. Recently, large-scale outbreaks suc-
(H1N1) virus during the recent epidemic, compared to related cessively swept through eastern Africa, the western Indian
swine influenza virus sequences (56). In further support of this Ocean islands, India, and southeastern Asia and also reached
hypothesis, the Indian Ocean epidemic sequences also exhib- Australia and Europe. These epidemics demonstrate the threat
ited an elevated number of nonsynonymous changes on exter- of this reemerging arbovirus and indicate the need to better
nal branches of the tree, reflected in a ratio of internal/external understand its evolutionary history and patterns and, particu-
dN/dS values of 0.97, compared to the lower values observed in larly, whether lineages differ in transmission cycles and ecolog-
other groups (0.53 to 0.63; Table 2). In theory, transient del- ical conditions, which could affect emergence potential.
eterious mutations are more likely to fall in the external Previous studies suggested a likely African origin of CHIKV
branches of trees (because they are short-lived), leading to a (45), where the virus circulates in an enzootic cycle between
higher dN/dS value than seen for internal branches. Although forest-dwelling Aedes species mosquitoes and nonhuman pri-
dN/dS ratios have limited utility in identifying positively se- mates. However, due to the deep divergence of the WAf and

TABLE 2. Selection pressures acting on each lineage of chikungunya virus

Mean dN/dS (95% CI) No. of negatively Positively selected
Data set I/Ea
Overall Internal External selected codonsb codon positionb

Asian21 0.125 (0.108–0.142) 0.095 (0.073–0.118) 0.152 (0.125–0.179) 0.63 70 NA

WAf11 0.066 (0.051–0.082) 0.040 (0.016–0.063) 0.075 (0.056–0.095) 0.53 64 NA
ECSA9 0.088 (0.060–0.115) 0.058 (0.049–0.068) 0.103 (0.080–0.126) 0.57 96 NA
Epidemic39 0.285 (0.232–0.338) 0.278 (0.056–0.497) 0.286 (0.300–0.343) 0.97 16 C-23; C-27; E1-226
a
I/E/, ration of internal to external values.
b
Significant at ␣ ⫽ 0.05.
6502 VOLK ET AL. J. VIROL.

ECSA lineages and the wide distribution in both East and evidence for differences in either mutation or replication rates
West Africa of the closest relative, ONNV, it is unclear in among CHIKV lineages, we propose that the differences in
which part of Africa CHIKV first evolved and, more intrigu- evolutionary rate between the enzootic and epidemic lineages
ingly, what causes the nearly complete transmission barrier are more likely due to the different transmission rates (as
that separates evolution in the two geographic regions. This discussed below), which affect the total amount of replication
question deserves further study given that (i) there are no per unit time, and/or differences in selection pressure.
obvious geographical barriers or ecological differences and (ii) The replication of alphaviruses in mosquitoes depends ini-
the two regions share the similar enzootic vector fauna and tially on ambient temperature (reviewed in reference 22) and

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potential primate reservoirs and (iii) given the occasional but is eventually modulated by poorly understood mosquito fac-
apparently nonpersistent mixing observed in the present study tors, including RNA interference (50). The resulting RNA
(the presence in Senegal of the ECSA clade, isolated from replication shutdown presumably leads to virtual genetic stasis
bats). despite the continued ability of infected vectors to transmit.
Epidemic origins. Interestingly, the Asian lineage dating to Therefore, the transmission rate directly influences the amount
ca. 1952 exhibits similar patterns of spread to the recent Indian of viral replication and consequently the numbers of mutations
Ocean outbreak lineage, with successive epidemics detected produced during the transmission cycle. The differing dN/dS
along an eastward path. This similarity suggests that the cur- values we estimated among epidemic versus enzootic CHIKV
rent Asian lineage may have also originated from an intercon- lineages, which may reflect differences in mildly deleterious
tinental transmission. Indeed, the tMRCA of the old Asian mutations sampled intensively during the recent CHIKV epi-
lineage (95% HPD: 1948 to 1956) almost immediately predates demics, could partially explain the higher evolutionary rate of
the Thailand outbreak of 1958, the first confirmed CHIK ep- this 2004 to 2008 epidemic lineage. However, different trans-
idemic in Asia. However, our current samples do not reveal mission patterns between the epidemic and enzootic cycles
exactly where the progenitor of the 1953 Asian outbreak oc- may be critical in regulating evolutionary rates in the two cycles
curred, although it was probably an ECSA strain that diverged (as illustrated in Fig. 3). In the relatively stable epidemic cycle,
from the currently circulating and sampled ECSA strains in the CHIKV is transmitted among humans via abundant perido-
beginning of the last century (95% HPD: 1879 to 1927). mestic mosquitoes, A. aegypti and A. albopictus, which colonize
The repeated CHIKV outbreaks that have occurred in India artificial and natural water containers in suburban and rural
and Southeast Asia are particularly noteworthy. In addition to areas (53). In contrast, transmission in the enzootic cycle dif-
epidemics corresponding to the Asian and Indian Ocean lin- fers dramatically between wet and dry seasons, with fluctua-
eages, CHIKV outbreaks probably have occurred in Asia for tions in vector densities associated with rainfall. During the dry
more than 200 years (10). These outbreaks were characterized season, due to the low vector population sizes, horizontal
by rapid progression, affecting hundreds to millions of people, transmission of CHIKV is probably maintained at a low level,
and were followed by long, silent interepidemic periods. Inter- whereas transovarial transmission may play a role in CHIKV
estingly, CHIKV apparently did not persist in these areas, with maintenance, as shown for other arboviruses (7). In addition,
the new outbreaks usually caused by introduction of imported lower vertebrate host availability in the enzootic cycle, where
strains, probably on sailing ships. The permanent Asian estab- primate populations are less dense than are humans in most
lishment of CHIKV during the 1950s may be attributed to the urban habitats, may also lead to a lower transmission rate.
increased human urbanization and expansion of A. aegypti pop- Moreover, greater herd immunity of reservoir hosts in the
ulations after World War II (21). The succession of CHIKV enzootic cycle could also reduce transmission efficiency.
outbreaks in India and Southeast Asia is likely related to the It is also possible that the lower evolutionary rates in enzo-
decline of herd immunity in human populations, as shown by otic lineages reflect stronger purifying selection, as suggested
the long epidemic interval (e.g., 33 years since the last Indian by the lower dN/dS values in the enzootic compared to the
circulation). In contrast, CHIK in Africa is endemic/enzootic, epidemic lineages (Table 2), which may in turn be related to
associated with limited, sporadic outbreaks (46), probably due the more diverse hosts and vectors used in the enzootic trans-
to the lower densities of human populations and their relative mission cycle. Enzootic CHIKV probably circulates in several
isolation in many cases, as well as more stable herd immunity different nonhuman primates, and possibly in bats or other
from periodic enzootic spillover. mammals, and is probably transmitted by several different
Variation in evolutionary patterns. We hypothesized that Aedes mosquitoes in the Celia subgenus (shown in Fig. 2) (16).
the difference in vectors and amplification hosts between the These diverse hosts and vectors may constrain the evolution
epidemic and enzootic transmission cycles may influence and adaptation of enzootic CHIKV compared to the epidemic
CHIKV evolutionary rates, which was supported by our data. lineages that rely only on humans and 2 closely related Aedes
Although a previous analysis of evolutionary rates in DENV-2 (Stegomyia) vectors. Another possibility is that epidemic trans-
virus, which also has ancestral enzootic and derived epidemic mission involves more CHIKV population bottlenecks if hu-
lineages, did not reveal faster evolution in the latter cycle, the mans generate lower viremia than nonhuman primates, leading
number of enzootic DENV strains analyzed in this case was to smaller numbers of virions establishing infection of the
very small, with resultant broad 95% HPD values (60). vector. However, if A. aegypti and/or A. albopictus are less
A variety of factors could contribute to the variation in susceptible than enzootic vectors, smaller numbers of virions
evolutionary rate that we observed for CHIKV, involving dif- may infect the midgut. Similar effects could occur if different
ferences in intrinsic factors, such as the rates of mutation and amounts of virus are transmitted in the saliva of epidemic
replication, or extrinsic factors, such as the strength of natural versus enzootic vectors. These bottlenecks could accelerate
selection or population transmission rates. Because there is no rates of sequence change by allowing the fixation of slightly
VOL. 84, 2010 CHIKV EVOLUTION IN TWO TRANSMISSION CYCLES 6503

The funding sources had no role in study design, data collection and
Enzootic Circulation 1 transmission
- 7 days high rep
analysis, decision to publish, or preparation of the manuscript.
- 18 days low rep
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JOURNAL OF VIROLOGY, June 2011, p. 5706 Vol. 85, No. 11
0022-538X/11/$12.00 doi:10.1128/JVI.00628-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.

AUTHOR’S CORRECTION
Genome-Scale Phylogenetic Analyses of Chikungunya Virus Reveal Independent
Emergences of Recent Epidemics and Various Evolutionary Rates
Sara M. Volk, Rubing Chen, Konstantin A. Tsetsarkin, A. Paige Adams, Tzintzuni I. Garcia,
Amadou A. Sall, Farooq Nasar, Amy J. Schuh, Edward C. Holmes, Stephen Higgs,
Payal D. Maharaj, Aaron C. Brault, and Scott C. Weaver
Center for Biodefense and Emerging Infectious Diseases and Department of Pathology, and Department of Biochemistry and
Molecular Biology, University of Texas Medical Branch, Galveston, Texas; Institut Pasteur de Dakar, Dakar, Senegal;
Center for Infectious Disease Dynamics, Department of Biology, Pennsylvania State University, University Park,
Pennsylvania; and Center for Vector-Borne Diseases and Department of Pathology, Microbiology and
Immunology, School of Veterinary Medicine, University of California, Davis, California

Volume 84, no. 13, p. 6497–6504, 2010. Supplemental Table 1: GenBank accession numbers of the sequences are HM04784 to
HM04823.

5706