Genotypic and Phenotypic Characterization of Chikungunya Virus of Different Genotypes from Malaysia

<div><h3>Background</h3><p>Mosquito-borne Chikungunya virus (CHIKV) has recently re-emerged globally. The epidemic East/Central/South African (ECSA) strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia.</p> <h3>Methods and Findings</h3><p>CHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and <em>Aedes</em> mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in <em>Ae. albopictus</em> cells (C6/36). Both CHIKV strains infected <em>Ae. albopictus</em> mosquitoes at a higher rate than <em>Ae. aegypti</em>, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected <em>Ae. aegypti</em> at higher rates.</p> <h3>Conclusions</h3><p>The greater ability of the ECSA strain to replicate in <em>Ae. albopictus</em> may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where <em>Ae. albopictus</em> predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and <em>Aedes</em> species.</p> </div

Comparative infection, replication kinetics, and dissemination of each Malaysian CHIKV strain in <i>Ae. aegypti</i> (⧫, black bars) or <i>Ae. albopictus</i> (⋄, white bars).

<p>The CHIKV isolates MY/06/37348 (Asian) and MY/08/065 (ECSA) were used. Following ingestion with MY/06/37348, infection rates of midguts of mosquitoes (A), replication in midguts as measured by a TCID₅₀ titration assay with plotted means ± SD of triplicates (B), and dissemination rates in salivary glands of mosquitoes (C) were determined. Following ingestion with MY/08/065, midgut infection (D), midgut replication (E), and salivary gland dissemination (F) were measured. Asterisks indicate significant differences (*p<0.05, **p<0.01). The denominator used to calculate midgut infection rates was the number of mosquitoes sampled, and the denominator for dissemination rates was the number of midguts with detectable virus titre. Denominators are shown (n).</p

Amino acid differences between Malaysian Chikungunya strains of Asian and East/Central/South African genotypes.

<p>Amino acid differences are reported following alignment of sequences obtained in this study from the Bagan Panchor strains MY/06/37348 and MY/06/37350 (Asian genotype), and the Johor strains MY/08/065 and MY/08/068 (ECSA genotype). The first amino acid named is found in the Asian strains, while the second amino acid is found in the ECSA strains. Differences at sites reported to be mosquito adaptation determinants in other alphaviruses are underlined.</p

Comparative replication kinetics of Malaysian CHIKV strains in cells.

<p>The CHIKV isolates MY/06/37348 (Asian, •) and MY/08/065 (ECSA, ○) were used. Replication was measured by virus titration using a TCID₅₀ assay in (A) Vero, (B) rhabdomyosarcoma, (C) C6/36, and (D) CCL-125 cells. Means ± SD of 3 independent experiments are plotted. Asterisks indicate significant differences (<i>p</i><0.05) at the same time-points.</p