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Project Topic
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Background: Ebolavirus and Marburgvirus ( hencefore denoted EBOV and MARV, respectively) are two genera of the negative sense RNA virus family Filoviridae, order mononegavirales. Filoviruses cause rare but fatal viral hemorrhagic fevers-VHF in equatorial Africa--with potential for regional and international urban spread. Both virus associated VHFs present with a similar prodrome; mimicking several tropical infectious diseases. Early detection is important for mobilizing swift response and control. Existing technologies for filovirus detection are not suited for point of care (POC) use, being expensive, not fastenough and requiring laboratory facilities absent in many village where index cases occur. While 2 rapid diagnostic tests (RDTs) have recently emerged detecting EBOV at the point of care (POC), there are no pan-filovirus targeted RDTs. Preliminary data: Our group—with prior funding from Grand Challenges Canada and now EDCTP, has synthesized, tested and validated monoclonal antibodies-mAbs and synthetic analogues of 3 conserved epitopes of filovirus glycoprotein (GP) for capture of (i) recombinant zaire EBOV GP ( cloned and expressed in HEK cells), and (ii) IgG antibody (in gamma irradiated serum of EVD survivors from the 2000 Gulu and Masindi outbreak, UVRI). However, none of the best performing sandwich EIAs (by capture of recombinant EBOV GP) detected native Gp on vero-expressed zaire EBOV virions, or patient samples in the P4 laboratory. Hypothesis: Post translational modifications of EBOV glycoproptein (GP) mask target epitopes from detection by mAbs. Objective (s): The overall goal of this project is to develop biochemical treatments that adjust native EBOV GP in patient sample as a target for rapid diagnostic testing. Four specific aims are contingent to this goal, viz: (i) pre-treatment with various mixtures ( namely a. glycosidases to remove glycans, b. endopeptidases to denature secondary structure, and c. reducing agents to break disulfide bond) to expose target conserved epitopes , (iii) Optimization of concentrations of buffer mixture (iv) ROC-characterization of the optimized sandwich EIA for filovirus rapid diagnostic testing, (iv) Prototype testing within the setting of the on-going EVD outbreak in Kivu, Democratic Republic of Congo-DRC. Approach: In-vitro experiments tailored to aims, within the P4 Lab at the CEZD, NICD, Johannesburg, SA and field testing on patient samples in Kivu, DRC. Potential Impact: This project could yield the 1st ever prototypes of RDTs for the duo-detection of EBOV and MARV. Pan-filovirus RDTs are required to ensure early detection, response and control of the ongoing and future outbreaks. Moreover, the mAbs presented are candidate therapeutics.
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