Chikungunya Virus Characterization and Development of Enzyme Linked Immunosorbent Assays as Detection Tools for Human and Mosquito Samples

Abstract

Chikungunya is a re-emerging disease which has become an important public health concern globally. In Kenya, there was a Chikungunya virus (CHIKV) outbreak in Lamu and Mombasa in 2004, which spread to the islands in the Indian Ocean then to South East Asia, Europe and America. Some of the challenges faced in this outbreak were due to lack of adequate surveillance and diagnostic tools to predict and facilitate early detection of the causative agent of the outbreak and the emergence of a more virulent strain of CHIKV mid-outbreak with enhanced transmission in a new mosquito species. To address some of these challenges, this study set out to characterize CHIKV from the 2005 CHIKV outbreak of Comoros Island and develop an Enzyme Linked Immunosorbent Assay (ELISA) as a diagnostic tool to detect CHIKV antigen in mosquito homogenates and anti-CHIKV antibodies in human sera. These tools would be developed in-house to ensure their availability and costeffectiveness. The CHIKV isolate from Comoros Island, was analyzed by plaque assay to quantify the viral titre. On observing the plaques, it showed plaques of different sizes, a large (L2) and small (S8) plaque. These plaques were purified and individual plaques infected culture fluid obtained and analysed by in vitro growth kinetics in different cell lines and their genetic similarity assessed by whole genome sequencing, comparative sequence alignment and phylogenetic analysis. Purified CHIKV antigen was used to immunize rabbits. The rabbit serum containing CHIKV specific polyclonal antibodies were purified and conjugated to horseradish peroxidase. An antigen detection ELISA was developed and evaluated using CHIKV positive and negative mosquito homogenates and the results confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). An in-house immunoglobulin M (IgM)-capture ELISA to detect CHIKV infections serologically was also developed using these reagents and compared with an independent IgM ELISA and a neutralization test using a panel of sera from the Comoros Island 2005 CHIKV outbreak. The in-house IgM-capture ELISA was used to test human sera samples collected during the 2013 Dengue outbreak in Kenya. Phenotypic and genetic characterization of the plaque variants showed higher viral titres of S7 compared to xxi L2 in C6/36 mosquito cell lines and a nonsense substitution in the nsp3 of S7, which was similar to a mutation in O`nyong nyong virus that had been shown to enhance infectivity and dissemination in Anopheles mosquitoes. This indicated the cocirculation of two variants with potentially different characteristics during the outbreak. The developed antigen detection ELISA used to test 48 mosquito pool homogenates showed a sensitivity (100%) and specificity (93.2%) when compared to the RT-PCR, with a kappa statistic of 0.70 indicating good agreement and can therefore be used as a surveillance tool for screening of CHIKV in mosquitoes. The IgM ELISA had a sensitivity (97.6%), specificity (86.9%) and a Cohen Kappa of 77% when compared to the Centers for Disease Control and Prevention IgM ELISA and a sensitivity (91.1%), specificity (96.7%) and Cohen Kappa of 88% when compared to a neutralization test. The assay was able to detect 26 CHIKV IgM positive out of 254 Dengue suspect human samples (10.2 %) indicating the utility of this assay in the field to detect Chikungunya co-circulating with other arboviruses. In conclusion, it was demonstrated that monitoring co-circulating strains of CHIKV by plaque typing is an effective and useful tool for the detection of emergent novel strains with potentially virulent phenotypes in mosquitoes and humans. Further studies are recommended using reverse genetics to confirm the effects of the identified amino acid substitutions on virulence in humans and vector competence in various mosquito species. The successful development of in-house assays for the detection of CHIKV antigen and antibodies has provided the tools for underresourced countries such as Kenya to conduct more robust diagnosis and surveillance for enhanced outbreak preparedness.