Molecular Characterization of Chikungunya Virus, Philippines, 2011–2013

During 2011–2013, a nationwide outbreak of chikungunya virus infection occurred in the Philippines. The Asian genotype was identified as the predominant genotype; sporadic cases of the East/Central/South African genotype were detected in Mindanao. Further monitoring is needed to define the transmission pattern of this virus in the Philippines.

conducted genetic analysis to characterize recent CHIKV infections that caused a large nationwide outbreak in the Philippines during 2011-2013.

The Study
Serum samples were collected through the chikungunya fever surveillance under the Philippine Integrated Disease Surveillance and Response of the Department of Health Epidemiology Bureau from different provinces in the Philippines during 2011-2013. Samples were collected from patients suspected to have chikungunya fever manifesting with symptoms such as fever, rash, and arthralgia. Samples were sent to the Research Institute for Tropical Medicine, which serves as the National Reference Laboratory for Dengue and Other Arboviruses. We screened serum samples by using CHIKV IgM-capture ELISA (NovaLisa, NovaTec Immundiagnostica GmbH, Dietzenbach, Germany). We extracted viral RNA using the QIAamp Viral RNA kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and amplified the partial E1 gene using 1-step reverse transcription PCR followed by direct Sanger sequencing with primers as previously described (10,11). We conducted phylogenetic analysis using the maximum-likelihood method as implemented in MEGA 6 software (http://www. megasoftware.net/). Molecular clock analysis and Bayes factor calculation were performed using BEAST software 1.8.0 (http://beast.bio.ed.ac.uk/) to select the best migration event model of CHIKV among countries. Bayes factor analysis was used to test phylogeographic hypothesis whether posterior migration rate between locations in whole evolutionary history was significantly higher than the expected prior migration rate, assuming truncated Poisson probability (12).
A total of 5,729 serum samples were collected from persons suspected to have chikungunya within 5 days after symptom onset. Fever, rash, and arthralgia were the most common symptoms (53%, 47%, and 34% of patients, respectively). Of the 5,729 serum samples, 2,891 were IgM positive by ELISA. We conducted reverse transcription PCR on 382 representative samples among IgM-negative patients in accordance with the CHIKV outbreak surveillance strategy of the Philippines, of which 131 samples tested positive. Partial E1 gene sequence (733 nt) was obtained from 31 samples. Sequences were submitted to Gen-Bank (accession nos. LC064714-LC064744).
Phylogenetic analysis identified 28 Asian genotype viruses and 3 ECSA genotype viruses (  analysis of E1 gene showed >99% nt similarity among 28 Asian genotype viruses (data not shown). Bayes factor analysis for the migration events also showed that migration might have occurred between Indonesia and Philippines with high probability (Table). CHIKV-positive patients were found only in Mindanao Island in 2011 but were detected in other parts of the country in 2012 and 2013. All CHIKV detected in parts of the country other than Mindanao were identified as Asian genotype. The 3 ECSA genotype viruses were detected in the Davao Del Sur and Davao Oriental in Mindanao Island in 2012 and 2013, and all were clustered into IOL (Figures 1,  2). This finding indicates that the 2 genotypes co-circulated in the island during the outbreak. ECSA genotype viruses in the Philippines were closely related to the Indonesian viruses (GenBank accession no. KC862329) detected in 2010. Sequencing analysis showed that all Philippine ECSA viruses possess the alanine to valine substitution (A226V) in the E1 gene (online Technical Appendix Tables 1, 2, http://wwwnc.cdc.gov/EID/article/22/5/ 15-1268-Techapp1.pdf).

Conclusions
The recent large CHIKV outbreaks in other Asian countries were caused mainly by the IOL of ECSA genotype (2). However, the 2011-2013 outbreak in the Philippines was caused mainly by the Asian genotype; the reason that this large outbreak was caused by this genotype is unknown. Previous reports have confirmed the reemergence of Asian genotype viruses in the Philippines (8,9). In this study, phylogenetic analysis and Bayes factor calculation showed that the Philippines viruses were closely related to Indonesian viruses, which might explain why the outbreak started in southern Mindanao, near Indonesia. Although sequence data of CHIKV in the database are not enough to identify the exact origin of the virus, we tried to estimate when CHIKV was introduced into the Philippines with molecular clock analysis using the dataset of Asian genotype viruses (online Technical Appendix Figures 1, 2). The results suggested that circulating Asian genotype viruses in the Philippines were introduced from Indonesia before 2010.
We also detected IOL of ECSA genotype, which possesses the E1-A226V substitution, and its co-circulation with the Asian genotype in the Philippines. However, the geographic distribution of ECSA genotype in 2012 and 2013 was limited to southern provinces in Mindanao. After the large outbreak in the Indian Ocean region in 2005, IOL of ECSA genotype rapidly spread to Asian countries and then co-circulated with the endemic Asian viruses, then eventually became the predominant genotype (13 (14) and in the Philippines in 2012 several years after the large outbreak in the Indian Ocean region (2). The movement of persons near the border of these countries might play a key role in CHIKV transmission. As part of the national vector control program, surveillance of Aedes aegypti and Ae. albopictus mosquitoes has been conducted in several areas in the Philippines. A previous report showed that the proportion of these 2 mosquito species was almost the same in Metro Manila (15). If the proportion of Ae. albopictus mosquitoes increases, the ESCA genotype virus with A226V mutation could spread more rapidly in the country. Thus, monitoring the spread of ECSA genotype viruses and the proportion of the Aedes mosquitoes in the Philippines is important.
We have demonstrated that the Asian genotype CHIKV, which is closely related to the Indonesian viruses, was identified in Mindanao in 2011 and spread to other regions in 2012 and 2013. Like the Asian genotype, ECSA genotype virus was first detected in Mindanao in 2012. Mindanao might play a key role for the introduction of the CHIKV into the Philippines. Further monitoring is necessary to define the transmission pattern of CHIKV, including cross-border transmission.