Genetic Divergence of Chikungunya virus from the Comoros Island (2005) and Detection of Chikungunya in a Dengue Outbreak Situation in Kenya in 2013 Caroline Wasonga 1, Shingo Inoue2, James Kimotho 3, Juliette Ongus 4 , Kouichi Morita2, Rosemary Sang 3, Lillian Musila5 University of Nairobi1, Nagasaki University2, Kenya Medical Research Institute3, Jomo Kenyatta University of Agriculture and Technology4, USAMRU-K5 Email: cwasonga@gmail.com/ cwasonga@uonbi.ac.ke; KENYA Logo Logo BACKGROUND INFORMATION AND INTRODUCTION AMINO ACID SUBSTITUTION AND PHYLOGENETIC ANALYSIS Chikungunya (CHIK) outbreak occurred in Lamu and Mombasa, Kenya in 2004. It spread to Comoros Island (2005-2006), and other islands in the Indian Ocean such as Reunion (2005), Mauritius (2005), Madagascar (2006), Seychelles (2006), and Maldives (2006). Then it further spread to India (2006), Sri Lanka (2006), Italy (2007), South Asia and Southeast Asian countries. In Reunion Island, a variant of chikungunya virus (CHIKV) (E1:A226V) that was transmitted by Aedes albopictus was reported. Emergence of variant viruses can have an impact on outbreaks and its analysis can give information about the route of epidemic spread by molecular epidemiological approach. Our CHIKV Comoros isolate (strain: Com5) showed large and small plaques in diameter and different patterns of growth kinetics. Sequencing analysis indicated several unique mutations. When CHIK outbreak was experienced in Kenya, diagnosis was delayed by lack of accurate diagnostics. To set up the CHIK diagnosis system in KEMRI, in-house IgM Capture ELISA (in-house ELISA) was developed by using locally prepared assay antigen and HRP conjugated anti-CHIKV rabbit IgG. Evaluation of in-house ELISA was done by comparison with CDC ELISA and focus reduction neutralization test (FRNT). This new in-house ELISA could successfully diagnose CHIK cases from the dengue outbreak patients in 2013 Amino acids (AA) alignment of Com5, L2, S7 and S27 strain, showed 30 AA changes in the non-structural protein (nsP) and 22 AA changes in the structural proteins. Between L2 and S7, a missense AA substitution of L2 in the nsp2, involving a conservative AA substitution and a nonsense substitution in the nsp3 of S7 which has been shown to enhance ONNV infectivity and dissemination in Anopheles mosquitoes was observed. Table 1a :Comparisons of amino acid sequences of the non-structural proteins of Com5, L2 and S7 and selected CHIKV Indian Ocean isolates using S27 prototype strain as a reference sequence Table1b: Comparisons of amino acid sequences of structural proteins of Com5, L2 and S7 and selected CHIKV Indian Ocean isolates using S27 prototype strain as a reference sequence.ference sequence Fig. 2 CHIKV parent strain and large and small variants Fig. 1 Map of CHIKV Cases in the Indian Ocean Islands OBJECTIVES To compare the biological characteristics between large plaque (L2) and small plaque (S7) of CHIKV Comoros5 strain. To develop in-house CHIKV IgM capture ELISA and validate it for setting up of a diagnosis system in KEMRI reference laboratory. Phylogenetic analysis showed that the parent strain and its variants clustered closely together with each other and with Indian Ocean CHIKV strains. METHODS Application to Dengue outbreak samples (2013) CHIKV propagation in Cell Culture Large-scale virus propagation and purification by ultracentrifugation Comparison with CDC-ELISA and FRNT IgG purification and HRP conjugation Antibodies generated in Rabbit Virus titration by plaque assay Plaque Purification of clones Whole genome sequencing of plaque clones In vitro growth kinetics of plaque clones Fig. 5.: Sequences of plaque variants obtained in this study indicated by () were compared with selected CHIKV strains from the Genbank. VALIDATION AND FIELD TESTING OF IN-HOUSE ELISA CHIK and DEN occur in the same environment and are transmitted by same vector.(Aedes sp).The in-house ELISA was used to screen samples from the DEN outbreak in Kenya in 2013 Table 2. Comparison of In-house IgM ELISA vs. FRNT Table 3: Distribution of CHIKV infection among in different districts in Kenya, 2013. Fig. 3. Flowchart of experiments RESULTS (a) Vero (b) C6/36 DISCUSSION Plaques of different sizes were observed in the plaque assays which suggest a co-circulating virus in the Comoros island isolate. In vitro growth kinetic studies show that the plaque variants are pure isolates and had higher viral titres in mosquito cells when compared to mammalian cells. The developed in-house ELISA is suitable for detecting anti-CHIKV antibodies in human serum for diagnosis purposes. There was circulation of CHIK during the Dengue outbreak that occurred in Kenya in 2013. ACKNOWLEDGEMENTS Fig 4. In vitro growth kinetics of the original CHIKV isolate (Com5) and the two plaque variants (L2 and S7) cultured in Vero (a) and C6/36 (b) cells Consortium for National Health Research funded the project and scholarship. USAMRU-K and KEMRI-CVR (VHF lab.) provided materials for the project. JICA-JSPS and JICA-JST SATREPS Projects provided equipment and Technical support. KEMRI Production Department and animal house provided facility for experiments. University of Nairobi offered study leave to enable this research to be done Jomo Kenyatta University of Agriculture and Technology offered training In vitro growth kinetics of parent strain(Com5), large (L2) and small (S7) plaques, were used to infect Vero and C6/36 cells. ICF were harvested daily for 3 days, and virus titres were determined by focus assay. All the three isolates had higher viral titres in C6/36 than in Vero cells (p = 0.03)