Development of an Enzyme-Linked Immunosorbent Assay to Determine the Expression Dynamics of Ebola Virus Soluble Glycoprotein during Infection

Abstract

Ebola virus (EBOV) is a highly pathogenic virus with human case fatality rates of up to 90%. EBOV uses transcriptional editing to express three different glycoproteins (GPs) from its GP gene: soluble GP (sGP), GP, and small sGP (ssGP). The molecular ratio of unedited to edited mRNA is about 70% (sGP): 25% (GP): 5% (ssGP), indicating that sGP is produced more abundantly than GP. While the presence of sGP has been confirmed in the blood during human EBOV infection, there is no report about its expression dynamics. In this study, we developed an EBOV-sGP-specific sandwich enzyme-linked immunosorbent assay (ELISA) using two different available antibodies and tested several animal serum samples to determine the concentration of sGP. EBOV-sGP was detected in nonhuman primate serum samples as early as 4 days after EBOV infection, correlating with RT-qPCR positivity. This ELISA might be further developed into a diagnostic tool for detection of EBOV in patients. Furthermore, this study provides insights into the expression dynamics of sGP during infection, which are important to decipher the function that sGP plays during infection.

The Ebola virus soluble glycoprotein contributes to viral pathogenesis by activating the MAP kinase signaling pathway

“…The system is well-established and has been used in previous studies investigating EBOV GP-mediated entry [ 7 ]. In addition, we produced recombinant EBOV and RESTV sGP in mammalian cells and concentrated it as described previously [ 16 ]. We infected HEK293 and Huh7 cells with VSV-EBOV GP in the presence of EBOV sGP, RESTV sGP, and a control protein (CP).…”

“…Further analysis of the effect of EBOV sGP on EBOV replication was performed using EBOV-sGP-KO, a recombinant EBOV with the GP gene mutated to not express sGP [ 16 ]. We confirmed the lack of sGP expression by WB using the mouse monoclonal antibody ZGP42/3.7, which detects both GP and sGP.…”

“…We previously established a sandwich ELISA to capture EBOV sGP. Using this assay, we did not detected any sGP in the supernatant of EBOV-sGP-KO-infected cells [ 16 ]. These results confirm that the GP mutation in the virus is stable and that there is no expression of sGP.…”

“…We followed an experimental strategy published by Strong and colleagues where mice were infected with EBOV and treated after 6 days with PMA [ 18 ]. In order to determine the optimal EBOV sGP concentration for treatment, we relied on previous reports that showed that EBOV sGP can be readily detected in the blood of EBOV-infected patients and animals in large quantities [ 16 , 26 ]. Based on this data, we determined that 200 μg are an appropriate concentration of sGP for the treatment EBOV-infected mice for 24 hours in order to determine if EBOV titers increase.…”

“…After the initial infection of target cells like dendritic cells and macrophages [ 27 ], sGP might facilitate and enhance initial spread in the early target tissue like the liver as shown here in Huh7 cells and mice. Later on, EBOV sGP accumulates in the serum at high concentrations [ 16 ] and plays a role in vascular dysfunction [ 28 ] and as an antibody decoy [ 15 ]. Future studies will expand upon this data set and will address outstanding questions like EBOV sGP concentrations in tissues over time during infection and the severity of pathogenicity of EBOV infection with and without sGP.…”