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Abstract
Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that produces an acute, usually non-fatal, febrile illness including Mayaro fever. Like other alphaviruses, the MAYV E1 and E2 envelope glycoproteins are major viral surface antigens that play a key role in host recognition and infection. Here, we report expression and purification methods for recombinant MAYV E1 (rE1) and rE2 using a baculovirus system. Enzyme-linked immunosorbent assays (ELISA) revealed that rE1 and rE2 were antigenic and reacted with human anti–MAYV IgG and IgM. Cross-reactivity was also confirmed with human anti-Chikungunya virus (CHIKV) IgG and IgM. Furthermore, we developed an immunochromatographic strip test (IST) with rE2 to diagnose MAYV infection. Thus, purified rE2 may be valuable tool for rapidly diagnosing MAYV infection.
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Fig. 1.
MAYV rE1 and rE2 expression using a recombinant baculovirus/insect system, (a) Schematic representation of MAYV genome organization, (b) Schematic of the pAcGP67A-E1 and pAcGP67A-E2 constructs generated. C-terminal His-tags were used to purify the MAYV E1 and E2 proteins, (c) Western blot analysis of media from pAcGP67A-E1- and pAcGP67A-E2-transfected Sf9 cells using anti-His-tag antibodies conjugated to horseradish peroxidase. Uninfected Sf9 cell cultivation media was used as a negative control (Neg. Ctrl.).
Fig. 2.
Analysis of MAYV rE1 and rE2 overexpression and purification by High Five™ cells, (a) SDS-PAGE of purified MAYV rE1 and rE2. The gel was stained with Coomassie brilliant blue R-250. (b) Western blot analysis of purified MAYV rE1 and rE2. After being transferred to a PVDF membrane, the proteins were detected by anti-His-tag antibodies conjugated to horseradish peroxidase. Uninfected High-Five™ insect cell cultivation media was used as the Neg. Ctrl.
Fig. 3.
rE1 and rE2 reactivity against human anti-MAYV IgG (a) and IgM (b). rE1 and rE2 cross-reactivity were determined by ELISA using anti-MAYV IgG and IgM or anti-CHIKV IgG and IgM. Each microplate well was coated with 100 ng of purified recombinant proteins. Bars indicate the mean absorbance at 450 nm. Error bars indicate standard deviations (SD) of triplicate measurements.
Fig. 4.
Schematic illustration of the MAYV IST assay (a, b) and an image of MAYV IST using rE2 to detect human anti-MAYV IgG and IgM (c). BSA was used as a Neg. Ctrl. Quantification of band intensity at the strip test line. Normalized pixel intensity was quantified using Image software. Intensity values were calculated relative to the control line intensity. The graph presents the ratio of signal to noise (S/N) on the test line.
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