Microneutralization assays were performed to detect neutralizing antibodies against CHIKV and ONNV in samples that were IgG‐positive for CHIKV VLP. Sera were diluted in a two‐fold series from 1:40 to 1:10 240 in PBS in a 96‐well plate. For each dilution, contact was performed with 100 plaque‐forming units (PFU) of CHIKV (BEI Resources: NR‐56523) or ONNV (BEI Resources: NR‐51661) for 1 h at 37°C in a 5% CO2 incubator. The complexes were then added to Vero cells (ATCC) CCL‐81, 3 × 104 cells per well. After 3–4 days of incubation, the cytopathogenic effects were investigated by 0.2% crystal violet (Sigma‐Aldrich) staining. After staining for 2–4 h, the plates were washed with copious amounts of tap water. For negative controls, at least three alphavirus‐naïve serum samples were used to incubate with CHIKV or ONNV before pipetting onto the Vero cells per plate. For positive controls, 100 PFU of CHIKV or ONNV without any contact with serum were placed on Vero cells in triplicate per plate.
The neutralizing titer was defined as the inverse of the highest dilution resulting in an infectious reduction of 50%. A titer lower than 1:40 was considered negative if cytopathogenic effects were not observed. Samples were considered positive if the titer was ≥ 1:40, that is, positive for ONNV if ONNV titers were at least two‐fold higher than CHIKV titers, and CHIKV positive if CHIKV titers were at least four‐fold higher than ONNV titers [32 (link)]. The threshold was lower for ONNV than CHIKV because of the unique one‐way cross‐reactivity between CHIKV and ONNV, that is, CHIKV antibodies are more likely to cross‐react with ONNV antigens than ONNV antibodies with CHIKV antigens [33 ]. All other results were interpreted as equivocal. Twenty Senegalese serum samples that were previously determined to be alphavirus‐naïve, were used to validate the assay. Each assay used at least four alphavirus‐naïve serum samples as negative controls.
The neutralizing titer was defined as the inverse of the highest dilution resulting in an infectious reduction of 50%. A titer lower than 1:40 was considered negative if cytopathogenic effects were not observed. Samples were considered positive if the titer was ≥ 1:40, that is, positive for ONNV if ONNV titers were at least two‐fold higher than CHIKV titers, and CHIKV positive if CHIKV titers were at least four‐fold higher than ONNV titers [32 (link)]. The threshold was lower for ONNV than CHIKV because of the unique one‐way cross‐reactivity between CHIKV and ONNV, that is, CHIKV antibodies are more likely to cross‐react with ONNV antigens than ONNV antibodies with CHIKV antigens [33 ]. All other results were interpreted as equivocal. Twenty Senegalese serum samples that were previously determined to be alphavirus‐naïve, were used to validate the assay. Each assay used at least four alphavirus‐naïve serum samples as negative controls.
