Emergence of ECSA-IOL E1-K211E/E2-V264A Lineage of Chikungunya virus during Malaysian 2021 outbreak

Abstract

Background: Chikungunya cases was reported to be on the rise in Malaysia from 2019 to 2021. Although potential endemicity was described previously, genotype shift during 2008 outbreak originating from the 2004 Indian Ocean Islands outbreak presents the probability of current CHIKV spread from neighboring countries. This is due to the prevalence of the new IOL sub-lineage which consists of E1-226A wildtype or reverted strains that are circulating in the Indian subcontinent before spreading to neighboring Thailand during 2018-2019 outbreak.

Method: In this study, samples received mostly from the Tangkak, Johor were analyzed. A total 56 CHIKV positive serum samples received in 2021 by Institute of Medical Research Malaysia (IMR), were collected based on sample selection criteria. Selected samples were subjected to total RNA extraction, whole-genome sequencing as well as bioinformatic analysis such as phylogenetic, variant and mutation analysis.

Results: Based on the genomic and phylogenetic analysis, the CHIKV samples from 2021 outbreak were of ECSA-IOL genotype. Genome similarity analysis also revealed that these CHIKVs were highly similar to 2018-2019 outbreak strain from Thailand. In comparison to the 2008 outbreak CHIKV isolate, the current CHIKVs lacked the E1-A226V mutation and harbored the new E1-K211E/E2-V264A sub-linage mutation. Since the E1-K211E/E2-V264A mutation facilitates adaptation to Ae. aegypti as opposed to the E1-A226V mutation which improves adaptation to Ae. albopictus, the emergence 2021 CHIKV outbreak in Malaysia can be postulated due to vector shift. Interestingly, a novel nsP3-T441A/V mutation detected in this study, may also play a role in virus transmission, pathogenicity, fitness and vector adaptation.

Conclusion: In summary, the current CHIKV outbreak are strains originated from the Indian subcontinent through Thailand which may have capitalized on vector shifting by adapting to Ae. aegypti. The presence of novel nsP3-T441A/V mutation may also contribute to the spread of this virus across peninsular Malaysia.

Keywords: CHIKV; Chikungunya; Malaysia; Sequencing; Whole-genome.

Fig. 1

Bar chart of Chikungunya cases in Malaysia from 2008 to 2021 based on based Malaysian Ministry of Health Press Statement on Dengue, Zika and Chikungunya fever situation from 2019 to 2021 [25, 26]. The chart shows the increasing Chikungunya cases from 2019–2021 since the last major 2008–2009 outbreak in Malaysia

Fig. 2

Genomic features annotation for sample IMR24-21 (11,810 bps) using Geneious Prime (version 2023.1.1) and Clone Manager 9

Fig. 3

Evolutionary analysis by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model. The tree with the highest log likelihood (-37,977.77) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 109 nucleotide sequences. There were a total of 12,359 positions in the final dataset. Evolutionary analyses were conducted in MEGA11

Fig. 4

Evolutionary analysis of IOL sub lineage by Maximum Clade Credibility (MCC) tree. The MCC tree involved 147 IOL CHIKV open reading frame from reference sequence and constructed by BEAST under uncorrelated lognormal clock and GTR + F + I + G4. The emergence of sub-lineages are with the time of the most recent common ancestor (tMRCA) and its 95% highest probability density (95% HPD) are indicated by black dots