Chikungunya Fever Outbreak, Bhutan, 2012

In 2012, chikungunya virus (CHIKV) was reported for the first time in Bhutan. IgM ELISA results were positive for 36/210 patient samples; PCR was positive for 32/81. Phylogenetic analyses confirmed that Bhutan CHIKV belongs to the East/Central/South African genotype. Appropriate responses to future outbreaks require a system of surveillance and improved laboratory capacity.

Six specimens, which were selected on the basis of their relatively strong CHIKV RT-PCR results, were used for partial sequencing of the envelope protein 1 (E1) gene; all 6 specimens were from Samtse.The 1,320-bp sequences were compared against 34 other GenBank E1 sequences from different localities (Figure 1).The 6 representative isolates (GenBank accession nos.KC731581-KC731586) were 99.9%-100% identical at the nucleotide level and 99.8% identical at the amino acid level.Phylogenetic analyses of the partial E1 sequences showed that the 2012 outbreak of chikungunya fever in Bhutan was caused by a CHIKV of the East/Central/South African genotype, and the virus was removed only by 0.5% from HM159388 India 2010 CHIKV, 0.7% from GQ229488 India 2009 CHIKV, and 0.5% from HM045801 Sri Lanka 2007 CHIKV.Amino acid sequence analyses showed that all 6 Bhutan isolates lack the E1 gene mutations, which led to the A226V E1 protein change found in the Réunion Island 06.49 strain and which are closely associated with a higher transmission rate (Figure 2) (10).
In addition, the 2012 Bhutan strain still retained the E211 amino acid, which is also found in the India 2010 strain (11).

Conclusions
The results of our phylogenetic analyses suggest that the Bhutan CHIKV originated from CHIKV found in India.This finding is not surprising given the proximity of the 2 countries and the expanding range of the virus.The 2012 chikungunya fever outbreak in Bhutan occurred in the same area as a 2004 outbreak of dengue virus infection (12); this area shares borders with West Bengal and Assam, India.
The IgM ELISA and RT-PCR results in our study did not always agree; however, the discrepancies may be explained by the time of collection relative to onset of symptoms.Specimens collected early after illness onset were likely collected during the viremic state and thus positive by RT-PCR, but not by IgM ELISA.Specimens collected later after illness onset would likely have been IgM positive, even in the absence of detectable virus.Because of several factors, the introduction of CHIKV virus into Bhutan was expected: the presence of the mosquito vectors (Ae.aegypti and Ae.albopictus) in southern districts, the increasing geographic reach of CHIKV, the predominance of a presumably naive population, urbanization and industrial growth in Bhutan, and the country's unrestricted border with India.Another contributing factor may have been the circulation of a new East/Central/South African CHIKV strain, first isolated in 2005 from a patient on Réunion Island, that represents a distinct clade within the larger East/Central/South African phylogroup (13).This clade has the A226V mutation in the E1 protein, which leads to higher transmission and replication in Ae. albopictus mosquitoes (10).However, isolates collected during this outbreak lack this mutation and instead are more closely associated with the India 2010 strain (Figure 2).
The 2004 dengue virus infection outbreak ( 12) and now the 2012 chikungunya fever outbreak in Bhutan suggest that despite its remote location, the country is still  vulnerable to epidemics and pandemics.A sound public health surveillance system and improved laboratory capacity is needed in Bhutan so that the country can appropriately respond to future disease outbreaks.
This work was supported by the Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance and Response System.Mr Wangchuk is chief of the Public Health Laboratory, Department of Public Health, Ministry of Health, Bhutan, and a member of National Committee for Immunization Practice.His research interests are infectious diseases.

Table 1 .
IgM ELISA results and signs and symptoms for patients with suspected chikungunya fever, Bhutan, 2012

Table 2 .
IgM ELISA and PCR results for 78 patients with suspected chikungunya fever, Bhutan, 2012* *NA, not applicable.†Results reported by the Public Health Laboratory, Department of Public Health, Ministry of Health, Thimphu, Bhutan.‡Results reported by the Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.§Mean age for female patients was 28.1 y; mean age for male patients was 26.5 y. ¶Mean age for female patients was 28.8 y; mean age for male patients was 28.7 y. ‖ Mean age for female patients was not reported; mean age for male patients was 31 y.