Emergence of Chikungunya Virus, Pakistan, 2016–2017

During December 2016–May 2017, an outbreak of chikungunya virus infection occurred across Pakistan. The East/Central/South African genotype was predominant. This study provides baseline data on the virus strain and emphasizes the need for active surveillance and implementation of preventive interventions to contain future outbreaks.

by using SPSS Statistics 16.0 (IBM, https://www.ibm. com). We predicted the atomic structure of the E1 protein by using the Semiliki Forest virus (PDB ID: 2XFC) as a model in UCSF Chimera version 1.11.2 (University of California, San Francisco, https://www.cgl. ucsf.edu/chimera).
We confirmed CHIKV by real-time reverse transcription PCR in 411 (70.3%) patients. The mean age of CHIKV-positive case-patients was 31.8 years (SD + 15.7 years); most (25.5%) were 21-40 years of age (Appendix Figure 1). Children ≤10 years of age had more rashes (42%) than adults, but 91% of patients 11-20 years of age and 40% of patients 21-30 years of age had joint pain and swelling.
Partial E1 (294 bp) and nsP1 (354 bp) gene sequences showed 99.9% similarity to strains of Indian Ocean lineage from the ECSA genotype ( Figure 2). CHIKV strains in Pakistan had 99.9% homology with viruses from India, Singapore, and Bangladesh. The E1 protein of isolates from Pakistan diverged only 0.01% from prototype S-27 from Africa.
We noted substitutions in E1 genes at T98A, S111T, A145T, and K157N (Table 2) and at K128T in the NSP1 genes. We analyzed potential glycosylation sites in E1 and compared these with an o'nyong-nyong virus strain (GenBank accession no. AF079456). The E1 gene revealed a single conserved N-linked glycosylation site at N141 (Appendix Figure 2).

Conclusions
We noted a high rate of CHIKV infection, 70% (411/ 584), among suspected cases; most, 37% (153/411), occurred during May, early in monsoon season, similar to a 1963 outbreak in India that coincided with monsoon season, July-December. Another study in India noted an increase in CHIKV infections during and after monsoon season, possibly reflecting the favorable breeding conditions for Ae. albopictus and Ae. aegypti mosquitoes (9).
With a population of >180 million, Pakistan is the world's sixth most populous country and the second most urbanized nation in South Asia; 36% of the population resides in cities. Pakistan and other countries in Asia are experiencing harsher summers and milder winters, conditions that increase outbreaks of arboviruses likely by expanding arthropod vector breeding seasons (10). In addition, outbreaks can intensify in poor sanitary conditions in parts of the region. CHIKV could also be introduced into nonendemic areas by travelers with viremia, leading to local transmission (11). Speculated risk factors in Sindh during 2016 included population movement across the country, as well as high vector density, poor sanitation, and susceptible populations (10).

308
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 2, February 2020 Genomic and serologic assays confirmed CHIKV infection 3-5 days after patients' fever onset. We noted more CHIKV cases in persons >20 years of age. However, persons <20 years of age more frequently exhibited rashes and arthralgia, similar to results from a previous study during a 2007 outbreak in Kerala, India (12).
CHIKV Asian genotype circulates in Southeast Asia, where Ae. albopictus mosquitoes have been expanding during the previous 60 years (13). In CHIVendemic settings in Asia, 2 independent E1 gene mutations in A226V and T98A could enable virus adaptation to this mosquito species. In contrast, for Indian Ocean lineage strains, the same fitness advantage and selection efficiency could be gained by the acquisition of a single T98A amino acid substitution (13).
Chikungunya should be considered as a diagnosis in persons who report fever, rash, or arthralgia, especially those returning from travel to virus-endemic areas (14). Chikungunya cases increased in Karachi and adjoining areas of Pakistan within months after massive outbreaks in India in 2016 (15). CHIKV in those outbreaks had high sequence homology to isolates from Pakistan, ascribing to the ECSA Indian Ocean lineage. Because no licensed CHIKV vaccine is available, public health officials should urge adoption of measures to prevent mosquito bites, such as use of repellents and mosquito nets.
Our study had some limitations. We did not obtain information on patient outcomes or clinical management. Our results might also underreport cases because Pakistan does not have a nationwide surveillance system for CHIKV.
In summary, we report on the molecular epidemiology of CHIKV genotypes circulating in Pakistan during 2016-2017. Sustained surveillance for CHIKV is needed to monitor the extent of virus circulation in subsequent years. Identification of genotypes and monitoring for mutations that might facilitate transmission fitness for CHIKV in mosquito vectors can improve public health response.
Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.