Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection

Abstract

In December of 2013, chikungunya virus (CHIKV), an alphavirus in the family Togaviridae, was introduced to the island of Saint Martin in the Caribbean, resulting in the first autochthonous cases reported in the Americas. As of January 2015, local and imported CHIKV has been reported in 50 American countries with over 1.1 million suspected cases. CHIKV causes a severe arthralgic disease for which there are no approved vaccines or therapeutics. Furthermore, the lack of a commercially available, sensitive, and affordable diagnostic assay limits surveillance and control efforts. To address this issue, we utilized an insect-specific alphavirus, Eilat virus (EILV), to develop a diagnostic antigen that does not require biosafety containment facilities to produce. We demonstrated that EILV/CHIKV replicates to high titers in insect cells and can be applied directly in enzyme-linked immunosorbent assays without inactivation, resulting in highly sensitive detection of recent and past CHIKV infection, and outperforming traditional antigen preparations.

Fig 1. Indirect mouse anti-CHIKV IgG ELISAs utilizing either (A) EILV/CHIKV or (B) cell-lysate antigen to detect serially diluted polyclonal anti-CHIKV MIAF (measured over a range of serum dilutions, red) or monoclonal antibody CHK-175 (expressed in ng quantities, blue).

MIAF against Gamboa virus and MIS against mosquito antigens were included as negative controls. Mean and standard deviation of 2 replicates are reported.

Fig 2. Human anti-CHIKV antibody-capture ELISAs utilizing EILV/CHIKV as antigen to detect either (A) IgM or (B) IgG antibodies (both measured over a range of serum dilutions, red).

Human serum samples that were antibody-positive for either DENV or VEEV but negative for CHIKV by HI were included as negative controls. Mean and standard deviations of 2 replicates are reported.

Fig 3. Comparison of EILV/CHIKV and Abcam human anti-CHIKV IgM-capture ELISA methods.

(A) raw OD values or (B) signal-to-noise ratios (calculated by dividing positive sample OD values by mean negative control OD value) produced by samples of varying degrees of seropositivity were compared. Low-positive samples had PRNT₅₀ ≤80 (n = 4), medium-positive samples had PRNT₅₀ 160–320 (n = 4), and high-positive samples had PRNT₅₀ >640 (n = 24). Mean and standard deviations are reported. A 2-way ANOVA with Tukey’s test was used to compare absorbance values and signal-to-noise ratios within each ELISA method. 2-way ANOVA with Bonferroni’s test was used to compare absorbance values and signal-to-noise ratios between each ELISA method.

Fig 4. Comparison of EILV/CHIKV and Abcam human anti-CHIKV IgG-capture ELISA methods.

(A) raw OD values or (B) signal-to-noise ratios (calculated by dividing positive sample OD values by mean negative control OD value) produced by samples of varying degrees of seropositivity were compared. Low-positive samples had PRNT₈₀ ≤80 (n = 10), medium-positive samples had PRNT₈₀ 160–320 (n = 10), and high-positive samples had PRNT₈₀ >640 (n = 10). Means and standard deviations are reported. A 2-way ANOVA with Tukey’s test was used to compare absorbance values and signal-to-noise ratios within each ELISA method. 2-way ANOVA with Bonferroni’s test was used to compare absorbance values and signal-to-noise ratios between each ELISA method.

Fig 5. Stability of EILV/CHIKV in different buffers determined by accelerated decay at elevated temperature.

Mean and standard deviations of three replicates are reported. A 2-way ANOVA with Tukey’s test was used to compare the effects of different buffer preparations on ELISA activity.

Fig 6. Safety of EILV/CHIKV in the newborn mouse model of neurovirulence.

(A) Survival of infant mice infected IC with EILV/CHIKV, live-attenuated vaccine strain 181/25, or PBS. (B) Replication of EILV/CHIKV or strain 181/25 in newborn mouse brain tissue.