Chikungunya Virus and the Mosquito Vector Aedes
aegypti in New Caledonia (South Pacific Region).
Myrielle Dupont-Rouzeyrol, Val´ erie Caro, Laurent Guillaumot, Marie Vazeille,
Eric D’Ortenzio, Jean-Michel Thiberge, No´ emie Baroux, Ann-Claire Gourinat,
Marc Grandadam, Anna-Bella Failloux
To cite this version:
Myrielle Dupont-Rouzeyrol, Val´ erie Caro, Laurent Guillaumot, Marie Vazeille, Eric D’Ortenzio,
et al.. Chikungunya Virus and the Mosquito Vector Aedes aegypti in New Caledonia (South
Pacific Region).. Vector-Borne and Zoonotic Diseases, Mary Ann Liebert, 2012, epub ahead of
print. <10.1089/vbz.2011.0937>. <pasteur-00764397>
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Chikungunya Virus and the Mosquito Vector
Aedes aegypti in New Caledonia (South Pacific Region)
Myrielle Dupont-Rouzeyrol,1Vale ´rie Caro,2Laurent Guillaumot,3Marie Vazeille,4
Eric D’Ortenzio,5Jean-Michel Thiberge,2Noe ´mie Baroux,5Ann-Claire Gourinat,6
Marc Grandadam,7,8,* and Anna-Bella Failloux4,*
Chikungunya virus (CHIKV) is transmitted to humans through the bite of Aedes mosquitoes. During the 2005–
2006 epidemic that occurred in the Indian Ocean Islands, a viral strain harboring a substitution of an alanine to
valine at position 226 (E1-A226V) of the E1 glycoprotein enhanced the transmissibility of CHIKV by Aedes
albopictus. In March 2011, autochthonous transmission of CHIKV was reported in New Caledonia (NC), an
island located in the southwest Pacific Ocean. This was the first report of local chikungunya (CHIK) trans-
mission in this region of the world. Phylogenetic analysis based on the complete genome demonstrated that the
CHIKV-NC strain isolated from the first autochthonous human case belongs to the Asian lineage. This is
consistent with the Indonesian origin of CHIK cases previously imported and detected. Thus the CHIKV-NC
does not present a valine substitution at position E1-226. In New Caledonia, the putative vector of CHIKV is
Aedes aegypti, since no other potential vector has ever been described. For example, A. albopictus is not found in
NC. Vector competence experiments showed that A. aegypti from New Caledonia was able to transmit, as early
as 3 days post-infection, two CHIKV strains: CHIKV-NC belonging to the Asian lineage, and CHIKV-RE from
Reunion Island harboring the E1-A226V mutation. Thus the extrinsic incubation period of both CHIKV strains in
this vector species could be considered to be quite short. These results illustrate the threat of the spread of
CHIKV in the South Pacific region. From February to June 2011 (the end of the alert), only 33 cases were
detected. Implementation of drastic vector control measures and the occurrence of the cold season probably
helped to limit the extent of the outbreak, but other factors may have also been involved and are discussed.
Key Words: Aedes aegypti—Arboviruses—Chikungunya—New Caledonia—Vector competence.
Togaviridae. CHIKV is transmitted to humans through the
bite of infected Aedes species mosquitoes, particularly Aedes
hikungunya fever is an acute illness caused by chi-
kungunya virus (CHIKV), an alphavirus of the family
aegypti and Aedes albopictus. It induces an arthro-febrile syn-
drome affecting mostly the extremities, and is associated with
polyarthritis and skin rash (reviewed in Schwartz and Albert
2010). The clinical illness is often associated with prolonged
morbidity, and significant economic and social consequences
1Institut Pasteur de Nouvelle-Cale ´donie, Re ´seau International des Instituts Pasteur, Laboratoire d’Epide ´miologie Mole ´culaire, Noume ´a,
2Institut Pasteur, Plate-forme Ge ´notypage des Pathoge `nes et Sante ´ Publique, Paris, France.
3Institut Pasteur de Nouvelle-Cale ´donie, Re ´seau International des Instituts Pasteur, Laboratoire d’Entomologie Me ´dicale, Noume ´a, New
4Institut Pasteur, Unite ´ de Ge ´ne ´tique Mole ´culaire des Bunyavirus, Paris, France.
5Institut Pasteur de Nouvelle-Cale ´donie, Re ´seau International des Instituts Pasteur, Unite ´ d’Epide ´miologie des Maladies Infectieuses,
Noume ´a, New Caledonia.
6Institut Pasteur de Nouvelle-Cale ´donie, Re ´seau International des Instituts Pasteur, Laboratoire de Diagnostic Spe ´cialise ´, Noume ´a, New
7Institut Pasteur, CNR des Arbovirus, Paris, France.
8Institut Pasteur of Lao PDR, Institut Pasteur International Network, Ventiane, Lao PDR.
*These authors contributed equally to this study.
VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 12, Number XX, 2012
ª Mary Ann Liebert, Inc.
described mainly in Africa and Asia, but the disease has re-
cently emerged in other parts of the world after carriage by
infected travelers, and CHIK is becoming a worldwide public
health problem. Phylogenetic analyses have demonstrated
three distinct lineages of CHIKV strains: West Africa, Asia,
and East/Central/South Africa (ECSA). In 2005 and 2006, the
Indian Ocean region was hit by an extraordinarily large
CHIK epidemic that reached India and Southeast Asia
(Hapuarachchi et al. 2010; Volk et al. 2010). The circulating
CHIKV strains from the Indian Ocean islands are derived
an alanine to a valine at position 226 of the CHIKV E1 gly-
coprotein. This substitution has been demonstrated to favor
CHIKV transmission by Ae. albopictus, but not by Ae. aegypti
(Schuffenecker et al. 2006; Vazeille et al. 2007; Tsetsarkin et al.
2007; de Lamballerie et al. 2008). Since the 2005–2006 Indian
Ocean outbreak, CHIKV strains from the ECSA group har-
extensively implicated (reviewed in Schwartz and Albert
2010). Indeed in 2007 in Europe, the first outbreak of CHIK
occurred in northeastern Italy, and resulted in 205 human
cases. The CHIKV strain was assigned to the ECSA group
harboring the substitution E1-A226V. The vector incrimi-
nated was Ae. albopictus (Rezza et al. 2007). Thus unexpect-
edly, in September 2010, autochthonous cases of CHIK were
detected in southeastern France due to viral strains belonging
to the ECSA group, which presented an alanine in E1-226
(Grandadam et al. 2011). Ae. albopictus was the likely vector.
These two European CHIK episodes underscore the dissem-
ination potential of CHIK in Europe and in other countries
where Ae. albopictus is present (reviewed in Paupy et al. 2009).
Local CHIKV transmission has never been reported in the
South Pacific region. The first outbreak occurred in New Ca-
ledonia (NC) from February to June 2011 (Alibert et al. 2011).
In this study, we identified the genotype of a CHIKV strain
isolated from thefirst autochthonous case in NC,and assessed
itstransmissionefficiencybyAe.aegypti populations from NC.
Materials and Methods
New Caledonia is an island located in the Western Pacific
region between latitude 20? and 22? south, approximately
2000km northeast of Sydney, Australia. The main city is
of the 250,000 New Caledonians live in Noumea and its sur-
rounding area. NC has a tropical climate with two marked
seasons: December to March being warm and wet (monthly
mean temperature between 26 and 27?C), and June to Sep-
tember being dry and much cooler (monthly mean tempera-
ture between 19 and 21?C).
Laboratory diagnosis and case definitions
Serum samples collected after 5 days following the onset of
illness were tested for IgM antibody to CHIKV with IgM im-
munocapture assays (MAC-ELISA; Pastorino et al. 2004). An-
tigens for those tests were prepared by the French National
Reference Centre for Arboviruses, Institut Pasteur, Paris,
France (CNR des Arbovirus). For IgM-positive cases, conva-
lescent sera was collected 2 weeks after the first sampling and
analyzed for CHIKV-specific IgG antibodies by an ELISA de-
veloped at the CNR des Arbovirus. RT-PCR detection of the
CHIKV genome was performed on blood during the first 6
days of infection, using the Superscript One-Step RT-PCR with
platinum Taq kit (Invitrogen, Carlsbad, CA; Pastorino et al.
2005), on LC480 (Roche Diagnostics, Mannheim, Germany),
after RNA extraction with the EasyMag system (bioMe ´rieux,
Paris, France). Considering the possibility of clinical confusion
with other viruses belonging to the Semliki Forest complex
present in the area, the first early sera were also tested for Ross
River virus (RRV) by real-time RT-PCR (Hall et al. 2010).
Serum samples were also tested for dengue virus infection
using IgM antibody capture ELISA (PanBio, Brisbane, Aus-
tralia), and NS1 Platelia antigen detection (BioRad, Marnes-
A clinically supported case was defined as a patient pre-
senting with sustained fever associated with arthralgia or ar-
viral RNA by RT-PCR, or a seroconversion or a significant rise
(fourfold) in antibody level. A probable case was defined as a
patient positive for virus-specific IgM antibodies in a single
serum sample collected at the acute or convalescent stage.
Origin of the New Caledonian CHIKV strain studied
The first two cases were serologically diagnosed and re-
lated to individuals returning from Indonesia at the begin-
ning of February2011. The first case (patient A), diagnosed on
February 25, 2011, lives in rural NC and did not cause any
secondary cases. The second case (patient B), identified after
interviewing patient A, lives in Noumea (Alibert et al. 2011).
Patient C, diagnosed on March 3, 2011 using RT-PCR (RRV
RT-PCR negative and dengue NS1 negative), lives near pa-
tient B, and was the first autochthonous CHIK case from
whom the CHIKV was cultured and analyzed. By late June
2011, 33 confirmed cases had been investigated by the health
authorities (Alibert et al. 2011). Virus transmission was ap-
parently interrupted after this date.
Viral genomic RNA was extracted from CHIKV grown once
on mosquito C6/36 cells. RT-PCR was performed using Super-
Script One-Step RT-PCR with platinum Taq (Invitrogen), with
primers targeting the complete genome (Schuffenecker et al.
2006). RT-PCR fragments were purified by ultrafiltration. Se-
quencing reactions were performed using the Big Dye Termi-
on automated sequence analyzer ABI3730XL (Applied Biosys-
tems). For sequence analysis, contig assembly and sequence
alignments were performed using program BioNumerics ver-
sion 6.5 (Applied-Maths, Sint-Martens-Latem, Belgium). For
phylogenetic analysis, a maximum-likelihood tree was con-
structed using MEGA version 5 software (www.megasoftware
.net), based on the Tamura-Nei model. The reliability of nodes
was assessed by bootstrap resampling with 1000 replicates.
Vector control during the outbreak
and entomological investigation
The day after the report of the first cases, intensive vector
control measures were implemented. These measures in-
cluded thorough and repeated source reduction carried out
2DUPONT-ROUZEYROL ET AL.
by city council staff, in addition to adulticide sprayings in the
dwellings of confirmed cases and within a 200-m radius.
Despite these measures, other cases were confirmed in the
residential district of patients B and C, and later the disease
spread to other areas of Noumea and the neighboring town-
ship of Dumbea, where similar vector control measures were
also implemented (Alibert et al. 2011).
Ae. aegypti populations were collected at immature stages in
March 2011 in two areas of Noumea, one close to the first
transmission cluster, and the second in another district. Mos-
quitoes were reared in a laboratory to obtain eggs for further
experiments. Asa routine measure carried out in the context of
vector surveillance, susceptibility of the two mosquito popu-
lations to deltamethrin was tested following the World Health
Organization (WHO) standard impregnated paper protocol
(World Health Organization 1981). A resistant strain had pre-
viously been established in the laboratory from a sample col-
lected in 2010, and was subsequently selected for resistance by
exposing individuals three times to the insecticide. The delta-
resistance, as suggested by Rivero and associates (2010).
Vector competence experiments
First-generation (F1) individuals were used for vector com-
petence experiments using two CHIKV strains displaying dif-
ferent genetic patterns and different potentials of transmission
by Ae. albopictus (Tsetsarkin et al. 2007; Vazeille et al. 2007):
CHIKV-NC, presenting an alanine residue at E1-226, and
CHIKV-RE from Reunion Island (2005), harboring the muta-
tion A226V in the E1 glycoprotein (named 06.21 in Schuffe-
necker et al. 2006). F1 females were allowed to feed on an
infectious blood meal at a titer of 107pfu/mL provided in a
glass feeder maintained at 37?C. The infectious meal was
composed of a virus suspension diluted (1:3) in washed rabbit
erythrocytes (Vazeille-Falcoz et al. 1999). The titer used was
females were sorted on ice and incubated at 28?C as described
in Vazeille and associates (2007). This temperature is close to
the monthly average warm season temperature (i.e., January
NC. At days 3, 8, and 14 post-infection, saliva samples were
collected as described in Dubrulle and colleagues (2009), and
the titer was estimated by focus fluorescent assay on C6/36
Ae. albopictus cell culture. The transmission rate corresponds to
the proportion of mosquitoes with infectious saliva (Dubrulle
et al. 2009; Vazeille et al. 2010).
Descriptive statistics consisted of the calculation of me-
dian, minima, and maxima for continuous variables, and
absolute numbers and proportions for categorical variables.
Comparative analyses were performed on the basis of the
Wilcoxon rank-sum test or the median test for continuous
variables, and Fisher’s exact test for categorical variables. A
p value<0.05 was considered statistically significant. Statis-
tical analyses were performed using Stata software version
11.0 (Stata Corporation, College Station, TX).
Bootstrap support values (1000 replicates) are indicated at major nodes. The scale bar indicates the number of base substi-
tutions per site (ECSA, East/Central/South Africa).
Phylogenetic relationships among several CHIK viruses based on complete genome (11,238 nucleotides) analysis.
CHIKUNGUNYA VIRUS AND A. aegypti IN NEW CALEDONIA3
Phylogenetic analysis of the New Caledonian
A molecular study of the CHIKV-NC strain (referenced as
NC/2011-568) obtained from patient C was conducted. Based
on the complete genome nucleotide sequence, phylogenetic
analysis demonstrated that the CHIKV-NC strain belonged
to the Asian lineage, which was consistent with the return from
Indonesia of patients A and B (Fig. 1). Furthermore, this isolate
Asian cluster. The amino acid sequence of CHIKV-NC revealed
the presence of an alanine at position E1-226, an aspartic acid at
position E2-60, and a threonine at positions E2-211 and E1-98.
The E1-98T residue corroborates the phylogenetic analysis, as it
has only been found in endemic Asian strains. It has been pro-
posed that E1-98T may restrict the ability of Asian CHIKV
strains to adapt to the vector Ae. albopictus (Tsetsarkin et al.
2011). The whole genome sequence of the CHIKV-NC strain
was deposited in GenBank under accession no. HE806461.
Mosquito investigation and susceptibility to pyrethroids
The putative vector of CHIKV in NC is Ae. aegypti, since no
other potential vector including Ae. albopictus has ever been
described (Lee et al. 1982; Guillaumot 2005). Despite com-
munity awareness campaigns and intensive vector control
carried out for many years in the context of dengue man-
agement, leading to a considerable decrease in vector density
during the last decade, the species is still well established,
particularly near human settlements. In 2011, relatively high
Ae. aegypti densities were reported in urban and peri-urban
areas of Noumea during the rainy season, from December to
April (Guillaumot unpublished data).
Insecticide resistance tests showed that the two field-
collected mosquito populations display a modified suscep-
tibility to deltamethrin with an average mortality of 88.5%
after 1h of exposure (Table 1). By contrast, the laboratory
strain is clearly resistant, with 58% mortality in the same
CHIKV competence of local A. aegypti
Vector competence refers to the biological ability of arthro-
pods to acquire, maintain, and transmit pathogens. No signif-
icant differences were found either in the transmission rate, or
in the number of viral particles in saliva, for the two CHIKV
strains among the three populations (Tables 2 and 3). No rela-
tion could be established between these results and suscepti-
resistance may have an impact on several characteristics of
vectors including vector competence (Rivero et al. 2010).
However, our results showed that local A. aegypti from
NC could transmit both CHIKV strains efficiently (Fig. 2). At
day 14 post-infection, the transmission rate of CHIKV-RE was
post-infection, the number of CHIKV-RE viral particles deliv-
ered in saliva was higher compared to CHIKV-NC (p=0.0485
and 0.019, respectively). With the CHIKV-NC strain, no sig-
nificant difference was found regarding the transmission rate
and the number of viral particles secreted on days 3, 8, and 14
post-infection in A. aegypti (p=0.348 and 0.979, respectively).
2009), we found that A. aegypti from NC was able to transmit
both CHIKV strains as early as 3 days post-infection. Thus, the
extrinsic incubation period (EIP) of both CHIKV strains in this
vector can be considered as very short.
We report here on the genotype of the CHIKV responsible
for the first autochthonous cases in NC, and show that local
A. aegypti populations are highly competent for this virus.
Despite this high competence level, no large outbreak oc-
curred in NC in a supposedly naive population (Alibert et al.
2011). Indeed, the short EIP obtained in the laboratory, and
high mosquito densities (Guillaumot unpublished data),
similar to those observed during the last dengue 4 episode in
2009, were probably sufficient to sustain an outbreak. How-
ever, as shown by the vector competence results, the NC
A. aegypti is more competent for CHIKV-RE from the ECSA
genotype than CHIKV-NC from the Asian genotype, with
higher transmission rates and viral loads in saliva. Even if the
Table 1. Characteristics of the Local
Aedes aegypti Populations Tested
Ae. aegypti laboratory
aMortality observed after 60min of exposure to papers impreg-
nated with 0.05% deltamethrin.
Table 2. Median Value of Viral Particles in Saliva Collected from Aedes aegypti
Pop 163/11Pop 174/11Pop 282/10
aMedian test. Infectious titer: 107pfu/mL.
CHIKV-NC corresponds to the strain NC 2011-568; CHIKV-RE corresponds to the strain 06.21 (harboring the mutation E1-A226V).
4DUPONT-ROUZEYROL ET AL.
CHIKV infectivity for A. aegypti was not influenced by the
E2-I211T mutation, the E2-G60D mutation was an impor-
tant determinant of CHIKV infectivity for A. aegypti (and
A. albopictus) (Tsetsarkin et al. 2009). As the CHIKV-NC and
CHIKV-RE strains used in this study were both harboring the
E2-G60D mutation, we were unable to draw an accurate
vector competence. Furthermore, at day 14 post-infection, the
number of viral particles secreted by A. aegypti infected with
the CHIKV-NC strain remained low. As the number of viral
particles necessary to efficiently infect a human is unknown,
this low number might be sufficient to contaminate and initi-
ate an outbreak. However, saliva collected from mosquitoes
infected with CHIKV-RE contains almost 10 times more viral
particles, with probable consequences for the disease severity
thatwasobserved on Reunion Island (Gerardinet al.2008). To
feed on blood, A. aegypti injects saliva during the intradermal
probing period, and is able to interrupt the intake of the blood
meal when disturbed. Thus multiple feedings may lead to
transmission of the virus to multiple hosts during the com-
pletion of one blood meal by a single mosquito (Platt et al.
1997). It is tempting to speculate that with a high viral load in
saliva, A. aegypti infected with CHIKV-RE is able to deliver
successive small quantities of infectious particles, while this
scenario is less applicable to CHIKV-NC.
Vector control measures and the beginning of the cold
season, combined with low transmission rates and low viral
loads in saliva, might have been sufficient to limit CHIKV
transmission. Other factors responsible for the emergence,
including ecological factors, social parameters, vector/host
associations, and host/virus genetic factors and their inter-
actions, should be thoroughly investigated. This study also
demonstrates that active foci of transmission or maintenance
of Asian genotype strains can still persist and spread in the
world via viremic travelers. Moreover, the potential to initiate
an outbreak from virus vertically acquired from infected fe-
males should be also investigated. Whereas the ECSA geno-
type has widely spread in the world since 2006, Asian isolates
still circulate and also represent a risk of emergence in regions
where competent vectors exist, and where the human popu-
lation is widely naive. As demonstrated during the recent
emergence in Europe (Rezza et al. 2007; Grandadam et al.
2011), imported cases returning from endemic countries rep-
resent a risk for CHIKV emergence. Thus surveillance and
diagnostic measures are essential to prevent this emergence.
The efficient CHIKV transmission that occurred in
NC highlights its potential dissemination throughout the
South Pacific region where it has never before been de-
scribed, although A. aegypti is ubiquitous and might play the
role of main vector. Even if the CHIKV transmission in NC
ceased during the southern winter, CHIK cases could be re-
introduced from Asia, and also from Reunion Island or Indian
Ocean Islands (via air travel), where the CHIKV-RE strain is
predominant. Thus the virus could propagate via viremic
travelers to other Pacific islands, as previously observed for
authorities in the Pacific community should pay attention to
other mosquito species present in the South Pacific region that
can act as efficient CHIKV vectors, such as A. albopictus and
other species belonging to the scutellaris group (Guillaumot
2005). Physicians should also be aware to test for CHIK when
patients present with dengue-like or influenza-like syndromes
associated with arthralgias without respiratory symptoms.
WethankSosiasi Kilama,OliviaO’Connor (InstitutPasteur
ofNewCaledonia), AnubisVega-Rua(Institut Pasteur,Paris),
and the virology diagnostic laboratory staff from the Institut
Table 3. Transmission Rate of Chikungunya Virus in Aedes aegypti
Pop 163/11Pop 174/11Pop 282/10
CHIKVn inf.n tot.% n inf.n tot.% n inf.n tot.%
aFisher exact test. Infectious titer: 107pfu/mL.
CHIKV-NC corresponds to the strain NC 2011-568; CHIKV-RE corresponds to the strain 06.21 (harboring the mutation E1-A226V).
n inf., number of infected females tested; n tot., total number of females tested.
the saliva of A. aegypti from NC after infection with the two
CHIKV strains. Transmission potential was evaluated by
estimating the number of infectious viral particles in excreted
saliva by fluorescent foci units on C6/36 cells. The trans-
mission rate is the ratio of the number of females positive for
CHIKV in their saliva per the number of females tested
(n=number of females tested).
Transmission rates and numbers of viral particles in
CHIKUNGUNYA VIRUS AND A. aegypti IN NEW CALEDONIA5
Pasteur of New Caledonia for technical assistance. We thank
Scott C. Weaver for providing the CHIKV sequences of the
Asian isolates used in this analysis. We are grateful to Su-
zanne Chanteau for scientific support and manuscript revi-
sion. We also thank Sylvia and Stephane Goiran and Anne
Barthel for manuscript revision.
This work was supported by Institut Pasteur (New Caledo-
nia and Paris), the Government of New Caledonia, and the
FrenchInstitut deVeilleSanitaire (InVS,SaintMaurice,France).
Author Disclosure Statement
No competing financial interests exist.
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6DUPONT-ROUZEYROL ET AL.