Expression and Purification of Recombinant Mayaro Virus Envelope Glycoproteins E1 and E2 to Develop a Mayaro Virus Detection System

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. and serological diagnosis using hemagglutination inhibition tests and enzyme-linked immunosorbent assays (ELISA); however, these methods are unresponsive for a great number of infectious samples and live virus analysis may require biosafety laboratories. Furthermore, due to the antigenic similarity of MAYV and CHIKV, there is an acute need to develop more specific and precise methods for diagnosing MAYV infection (9). The MAYV genome is a 12kbp long single-stranded RNA containing two open reading frames (ORFs). It encodes four non-structural proteins (nsP1, nsP2, nsP3, and nsP4) and five structural proteins (C, E3, E2, 6K, and E1). The structural envelope glycoproteins E1 and E2 are imbedded in the envelope on the viral surface. E1 mediates the fusion of the virus with the host cell, while E2 is mainly involved in attaching viruses to host cells (10). As for alphaviruses, E1 and E2 are targets of the anti-MAYV antibody response; therefore, E1 and E2 would be useful targets for immunodiagnostic analysis. In this study, we explain the generation of soluble MAYV E1 and E2 using a baculovirus/insect expression system. Recombinant envelope proteins E1 (rE1) and rE2 were successfully expressed and purified in soluble forms, with rE2 better expressed and more stable than rE. ELISA revealed that rE1 and rE2 were antigenic and reactive for specifically detecting human anti–MAYV IgG or IgM. Cross-reactivity was also evaluated using human anti–CHIKV IgG or IgM. In particular, rE2 displayed a higher ELISA value than rE1. Furthermore, we developed an immunochromatographic strip test (IST) using rE2 to detect human anti-MAYV IgG or IgM, demonstrating the potential applicability of rE2 in a diagnosis system to detect MAYV infection. at 25°C, washed six times with PBST, and incubated with HRP–conjugated goat–anti mouse IgG antibodies at a 1:10,000 dilution for 1 h at 25°C. To visualize antibodies bound to the proteins, ECL luminal kits were used as enzyme substrates and the blots were visualized on a chemiluminescence imaging system (WSE–6200H LuminoGraph II, ATTO Corp, Tokyo, Japan). rE1 and rE2 reactivity against human anti–MAYV IgG and IgM was tested (11). A 96-well microtiter plate (Costar 3690, Corning Inc., New York, USA) was coated with 100 ng/well of purified rE1 or rE2 diluted to 1 µ g/mL in 0.1 M carbonate– bicarbonate buffer (pH 9.0) overnight at 4°C. Unbound antigens were discarded and the wells were blocked with 2% BSA in PBS for 60 min at 25°C. After washing, the wells were incubated at 37°C for 2 h with MAYV positive controls (human anti–MAYV IgG or IgM) from an anti–MAYV ELISA kit (Euroimmun, Luebeck, Germany). CHIKV positive controls (human anti–CHIKV IgG or IgM) from an anti-CHIKV ELISA kit (Euroimmun, Luebeck, Germany) were used to test cross-reactivity. The plates were washed six times with PBST and incubated at 37°C for 1 h with HRP-conjugated anti–human IgG or IgM diluted at 1:10,000. Bound antibodies were detected by adding 3, 3′, 5, 5′–tetramethylbenzidine (TMB, Sigma, St. Louis, Missouri, USA) and optical density was measured at 450 nm using an ELISA microplate reader (Thermo Scienti fi c, Waltham, Massachusetts, USA). Bars represent the average absorbance of three measurements and the error bars indicate standard deviations (SD) of triplicate measurements.