Reemergence of Chikungunya virus in Cambodia.
Subject: Disease transmission (Genetic aspects)
Disease transmission (Health aspects)
Authors: Duong, Veasna
Andries, Anne-Claire
Ngan, Chantha
Sok, Touch
Richner, Beat
Asgari-Jirhandeh, Nima
Bjorge, Steve
Huy, Rekol
Ly, Sovann
Laurent, Denis
Hok, Bunheng
Roces, Maria Concepcion
Ong, Sivuth
Char, Meng Chuor
Deubel, Vincent
Tarantola, Arnaud
Buchy, Philippe
Pub Date: 12/01/2012
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: Dec, 2012 Source Volume: 18 Source Issue: 12
Accession Number: 313345665
Full Text: Chikungunya virus (CHIKV; family Togaviridae; genus Alphavirus) is an arthropod-borne virus transmitted to humans by Aedes spp. mosquitoes (1). It is an enveloped, positive-sense, single-stranded RNA virus with a genome of -11.8 kb (2). Three genotypes have been identified: Western African, East Central South African (ECSA), and Asian (3).

First identified in Tanzania in the mid-1950s, CHIKV circulated in the 1960s in sub-Saharan Africa and several Asian countries (4,5). Reemergence of CHIKV (ECSA genotype) was reported in Democratic Republic of Congo in 1999-2000 (6,7) and in Kenya in 2004 (4). This genotype emerged in Comoros followed by Reunion Island, the Seychelles, Mauritius, Mayotte, and India in 2005 (the Indian Ocean outbreak); in Sri Lanka and Malaysia in 2006; in Singapore and Thailand in 2008; and in China in 2010 (8).

In Cambodia, CHIKV was first detected in 1961, probably the Asian genotype that was circulating in the region at that time (9). Since 2000, all blood specimens collected by the National Dengue Control Program, Ministry of Health Cambodia, during hospital-based surveillance of dengue and investigation of suspected dengue cases have been screened for IgM against CHIKV, dengue virus (DENV), Japanese encephalitis virus (JEV), and other arboviruses. Despite this testing, no evidence of acute or recent CHIKV infections has been found. To confirm CHIKV infection in samples positive by serologic testing, we conducted realtime reverse transcription PCR (RT-PCR) and complete genome sequencing of the samples. In 2011, we detected CHIKV ECSA genotype in patients in Cambodia and analyzed the phylogenetic origin of the strains.

The Study

We obtained samples from 3 sources: national dengue-like surveillance, an encephalitis study, and the outbreak investigation. From surveillance, during 2000-2011, an average of >700 paired serum samples were collected annually from patients admitted to sentinel hospitals for dengue-like syndrome (Battambang, Siem Reap, Kampong Cham, Takeo, Phnom Penh; Figure 1) in the National Dengue Control Program (10). From the encephalitis study, conducted July 2010 through July 2011, samples from 196 patients were collected as part of a surveillance study of central nervous system infections in Jayavarman VII hospital in Siem Reap (with written consent from patients or legal guardians and study approval by the National Ethics Committee in Cambodia). During the outbreak investigation, serum was collected during investigations by National Health authorities in Preah Vihear Province on August 16 (n = 9) and December 9-10, 2011 (n = 8), of outbreaks of suspected measles-like or dengue-like illnesses (Table).

All serum was tested at the Institut Pasteur in Cambodia. Acute-phase and/or convalescent-phase specimens were tested for IgM against flaviviruses (DENV, JEV, and Langat virus) and alphaviruses (CHIKV Ross C 347 strain and Sindbis virus). We used in-house IgM-capture ELISA as described by Vong et al. (11) with JEV, Langat, Sindbis, DENV, and CHIKV antigens; CHIKV was isolated by use of a mosquito cell line (clone C6/36 of Aedes albopictus cells) (11). Viral RNA was extracted from 140 [micro]L of serum by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer's recommendations. The presence of CHIKV RNA was determined by real-time RT-PCR selective for the E1 gene, according to a protocol adapted from Pastorino et al. (12) for a different Taq polymerase kit (SuperScript III Platinum One-Step Quantitative RT-PCR kit; Invitrogen, Carlsbad, CA, USA). Each series of tests included a negative control.


Among 19 samples positive for CHIKV by real-time RT-PCR, 8 were selected for complete genome sequencing. A total of 22 overlapped PCR products were obtained by using primers published by Schuffenecker et al. (13) and sent to Macrogen (Seoul, South Korea) for sequencing.

Sequence assembly and alignment were performed by using the CLC Main Workbench 5.5 package (CLC bio A/S, Aarhus, Denmark). The complete coding region (11,319 nt) of 8 CHIKV isolates from Cambodia were aligned with 64 reference strains available in GenBank. Phylogenetic analysis was performed by using the maximum-likelihood approach incorporating the GTR+r4 model of nucleotide substitution with a bootstrap resampling process of 1,000 replications.

During this reemergence of CHIKV in 2011, a total of 24 patients with fever, sometimes associated with acute arthritis or encephalitis (suggesting that many classical infections were not reported because encephalitis is a rare complication of chikungunya), had positive RT-PCR and/ or IgM-capture ELISA results for CHIKV. The first 2 cases were identified in May 2011 by the National Dengue Control Program in Battambang Province (eastern Cambodia) near the Thailand border (Figure 1). These 2 cases were in children hospitalized for suspected dengue. Subsequent cases were reported the same year in Siem Reap (2 cases, June and July), Kampong Thom (1 case, July), Kampong Cham (1 case, October), and Kandal (1 case, December) Provinces. Two other outbreaks were documented in villages in Preah Vihear Province (northern Cambodia) in August (9 cases) and December (8 cases) 2011 (Table). The sequence of outbreaks, in time and space, suggests that the virus was introduced to areas bordering Thailand and progressed through Cambodia, affecting city and villages along major northwest to southeast routes (Figure 1). Average patient age was 20 years (range 2-56 years); cases were distributed equally among male and female patients.


The alignment of the E1 gene sequence of CHIKV showed that all 8 strains carried the same amino acid substitution in the E1 protein (E1-A226V) as did the strains that were naturally selected by the mosquito vector a few months after the beginning of the Indian Ocean outbreak (13). Phylogenetic analysis of the complete genome sequence revealed that all viruses from Cambodia clustered with those isolated during the Indian Ocean outbreak and within the ECSA phylogenetic group (Figure 2). These isolates from Cambodia were closely related to the viruses isolated from southern Thailand during the 2008-2009 outbreak with bootstrap values <70 (data not shown) and to other isolates from the recent outbreaks in Asia (Singapore, Malaysia, Indonesia, and China). The pairwise nucleotide comparison of the complete coding region showed a high average percentage of similarity (>99.50%) with the recent isolates from Thailand, Malaysia, Singapore, and China. The identity between the strains from Cambodia ranged from 99.89% to 99.93% at the nucleotide level. Of note, the Cambodian strains can be separated into 2 groups supported by a bootstrap value of 100, suggesting that the viruses isolated in Battambang and Preah Vihea Provinces, which each border Thailand, could have been introduced separately, although we cannot exclude the possibility of introduction from other Asian countries as well.

As numbers of reported cases, numbers of provinces affected, and abundance of mosquito vectors (Ae. aegypti and Ae. albopictus) increase, the risk for local transmission will probably increase in the next few years, and levels of CHIKV infection could reach those of DENV infection. The ECSA genotype could then become endemic to Cambodia, which could face the same situation as in the 1960s, when a number of chikungunya cases were reported in Cambodia, although the 1960s epidemic did not last long (according to data available) and was not followed by continuous virus circulation leading to successive outbreaks. As CHIKV reemerges after 50 years of absence or low-level transmission, cocirculation with DENV might cause substantial challenges for public health, especially hospital overloading and increased needs for case management. This outbreak of CHIKV ECSA genotype spread rapidly in Cambodia over a short 7-month period. The outbreak should serve as a warning for health authorities to prepare, not only in Cambodia, but also in other areas where, to our knowledge, this genotype has not been reported, such as Vietnam and Lao People's Democratic Republic.



We thank Mao Sok, Ky Santy, Yun Saophondara, Deng Ankirboss, and Eng Chaya, and the staff of the Technical Bureau of Preah Vihear Provincial Health Department, for providing samples, and we thank Ung Sam An and the staff from National Institute of Public Health Cambodia for their fruitful collaboration.

This work was supported by French Ministry of Research. The serosurveillance studies and outbreak response were funded by a World Health Organization grant to the Ministry of Health and Institut Pasteur in Cambodia in 2011. The World Health Organization acknowledges the Korea International Cooperation Agency and the World Bank for their generous support for these activities.

Dr Duong is a senior virologist at the Institut Pasteur in Cambodia. His research interests include arboviruses and emerging zoonotic viruses.

Author affiliations: Institut Pasteur in Cambodia, Phnom Penh, Cambodia (V. Duong, A.-C. Andries, S. Ong, V. Deubel, A. Tarantola, P. Buchy); Ministry of Health, Phnom Penh (C. Ngan, T. Sok, R. Huy, S. Ly, M.C. Char,); Jayavarman VII Hospital, Siem Reap, Cambodia (B. Richner, D. Laurent); World Health Organization, Phnom Penh (N. Asgari-Jirhandeh, S. Bjorge, M.C. Roces); and Preah Vihear Provincial Health Department, Preah Vihear, Cambodia (B. Hok)

DOI: 10.3201/eid1812.120471


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Address for correspondence: Philippe Buchy, Institut Pasteur in Cambodia, Virology Unit, No. 5, PO Box 983, Monivong Blvd, Phnom Penh, Cambodia; email:

All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required.
Table. Characteristics of 24 patients with positive results for
chikungunya virus, Cambodia, 2011 *

Patient      Project            y/sex

V0603308      NDCP               2/M
V0603310      NDCP               4/F
V0705309       CNS               5/M

V0719309       CNS              14/M
V0719310       CNS               7/M
V1005304      NDCP              15/M
V1024306       CDC              11/F
V1024307       CDC              11/F
V1024308       CDC              28/F
V1024309       CDC              30/F
V1024310       CDC              17/M
V1024311       CDC              2.5/F
V1024312       CDC              13/F
V1024313       CDC              10/F
V1024314       CDC              31/F
V1214306       NMC              49/M
V1214307       NMC              56/M
V1214308       NMC              29/M
V1214309       NMC              39/F
V1214310       NMC              17/F
V1214311       NMC              27/M
V1214312       NMC              16/M
V1214333       NMC              30/F
V1207304      NDCP               9/F

             Date of
             symptom            Date
Patient       onset            sampled

V0603308     May 26            May 28
V0603310     May 26            May 28
V0705309     Jun 23            Jun 30

V0719309      Jul 9            Jul 17
V0719310     Jul 10            Jul 13
V1005304     Sep 30             Oct 4
V1024306     Aug 10            Aug 16
V1024307     Aug 15            Aug 16
V1024308      Aug 8            Aug 16
V1024309      Aug 8            Aug 16
V1024310     Aug 13            Aug 16
V1024311     Aug 16            Aug 16
V1024312     Aug 16            Aug 16
V1024313     Aug 14            Aug 16
V1024314     Aug 15            Aug 16
V1214306      Dec 9             Dec 9
V1214307      Dec 6             Dec 9
V1214308      Dec 5             Dec 9
V1214309      Dec 9            Dec 10
V1214310      Dec 9            Dec 10
V1214311      Dec 7            Dec 10
V1214312      Dec 9            Dec 10
V1214333       NA              Dec 10
V1207304      Dec 1             Dec 6

Patient     Province          reported

V0603308       BTB        Dengue ([dagger])
V0603310       BTB             Dengue
V0705309       SRP          Encephalitis
                          ([double dagger])
V0719309       SRP          Encephalitis
V0719310       KTH          Encephalitis
V1005304       KCH             Dengue
V1024306       PVH       Measles ([section])
V1024307       PVH             Measles
V1024308       PVH             Measles
V1024309       PVH             Measles
V1024310       PVH             Measles
V1024311       PVH             Measles
V1024312       PVH             Measles
V1024313       PVH             Measles
V1024314       PVH             Measles
V1214306       PVH             Dengue
V1214307       PVH             Dengue
V1214308       PVH             Dengue
V1214309       PVH             Dengue
V1214310       PVH             Dengue
V1214311       PVH             Dengue
V1214312       PVH             Dengue
V1214333       PVH             Dengue
V1207304       KAN             Dengue

Patient        1st               2nd

V0603308       Neg               Pos
V0603310       Neg               Pos
V0705309       Neg               Pos

V0719309       Pos               Pos
V0719310       Pos               Pos
V1005304       Neg               Pos
V1024306       Neg               NA
V1024307       Pos               NA
V1024308       Neg               NA
V1024309       Pos               NA
V1024310       Neg               NA
V1024311       Neg               NA
V1024312       Neg               NA
V1024313       Neg               NA
V1024314       Neg               NA
V1214306       Neg               NA
V1214307       Neg               NA
V1214308       Neg               NA
V1214309       Neg               NA
V1214310       Neg               NA
V1214311       Pos               NA
V1214312       Neg               NA
V1214333       Neg               NA
V1207304       Pos               Pos

Patient       Serum              SN

V0603308       Pos               Pos
V0603310       Pos               Pos
V0705309       Neg               Neg

V0719309       Neg               Neg
V0719310       Pos               Neg
V1005304       Pos               Pos
V1024306       Pos               Neg
V1024307       Neg               Neg
V1024308       Pos               Neg
V1024309       Pos               Pos
V1024310       Pos               Pos
V1024311       Pos               Pos
V1024312       Pos               Pos
V1024313       Pos               Pos
V1024314       Pos               Pos
V1214306       Pos               Neg
V1214307       Pos               Neg
V1214308       Pos               Pos
V1214309       Pos               Pos
V1214310       Pos               Pos
V1214311       Neg               Neg
V1214312       Pos               Pos
V1214333       Pos               Pos
V1207304       Neg               Neg

Patient    sequencing

V0603308     Partial
V0603310    Complete
V0705309       ND

V0719309       ND
V0719310       ND
V1005304       ND
V1024306    Complete
V1024307       ND
V1024308    Complete
V1024309       ND
V1024310    Complete
V1024311    Complete
V1024312       ND
V1024313    Complete
V1024314    Complete
V1214306       ND
V1214307       ND
V1214308       ND
V1214309       ND
V1214310       ND
V1214311       ND
V1214312       ND
V1214333       ND
V1207304       ND

* All patients had negative IgM-capture ELISA results for flaviviruses
(dengue 1-4, Japanese encephalitis, Langat) and other alphaviruses
(Sindbis). RT-PCR, real-time reverse transcription PCR; 1st,
acute-phase serum sample; 2nd, convalescent-phase serum sample;
SN, supernatant; NDCP: National Dengue Control Program,
National Malaria Center, Ministry of Health; BTB, Battambang;
Neg, negative; Pos, positive; CNS, central nervous system
infection (encephalitis) study; SRP: Siem Reap; ND, not
done; KTH: Kampong Thom; KCH: Kampong Cham; CDC, Communicable
Disease Control Department, Ministry of Health, Preah Vihear
outbreak investigation in August 2011; PVH: Preah Vihear;
NA, not available; NMC, National Malaria Center,
Ministry of Health, Preah Vihear outbreak
investigation in December 2011; KAN, Kandal.

([dagger]) Dengue-like: fever with headache, retro-orbital pain,
myalgia, joint pain, and/or hemorrhage.

([double dagger]) Encephalitis: fever with convulsion, vomiting,
and/or hemiplegia.

([section])Measle-like: fever with rash, cough, or join pain.
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