Investigating the role of Onchocerca volvulus virome and excretory-secretory products in the pathogenesis of Onchocerciasis-Associated Epilepsy

Recent evidence suggests that onchocerciasis is epidemiologically-linked to epilepsy, named Onchocerciasis-Associated Epilepsy (OAE). However, the underlying pathophysiological mechanism remains unknown. This research will focus on microfilaria from O. volvulus and OVRV1, an recently discovered virus of O. volvulus, and their potential roles in the pathogenesis of OAE.

Objective
The overall objective is to determine the potential pathogenic role of OVRV1 in OAE development. Specifically, I aim to
1) Investigate whether OVRV1 is present in the cerebrospinal fluid (CSF) of children with febrile seizures in onchocerciasis-endemic areas with high O. volvulus transmission;
2) Demonstrate whether OVRV1, O. volvulus mf and/or excretory-secretory proteins (ESP) can cross the BBB.

Research question 1
How to detect OVRV1 in CSF?
Method
I will optimize the detection of OVRV1 by designing a qPCR to detect OVRV1 in real-time and quantify the infection load. First, viral RNA will be isolated and converted to cDNA using commercially available kits. The qPCR will be designed based on a SYBR Green detection method and will be preceded by a conventional PCR utilizing nested primers to enhance specificity. The initial PCR will be performed on the generated cDNA using the primers developed by Quek et al(8). Afterwards, the resulting PCR product will be used as a template for the qPCR, for which I will design new primers. The qPCR assay will be validated by testing sequencing confirmed OVRV1 positive and negative samples. The detection limit will be determined using a standard curve of OVRV1 virus titers. Finally, both the PCR and qPCR products will be sequenced to ensure specificity.

Research question 2
Are O. volvulus biomarkers (OVRV1, O. volvulus mf DNA and/or ESPs) present in CSF of children with early onset OAE?
Method
Examination of CSF of children with complicated febrile seizures in an onchocerciasis-endemic areas
I will use aliquots of CSF samples that were collected from ivermectin-naïve children aged 2-5 years with complex febrile seizures, after obtaining informed consent from their parents. This study will be conducted in onchocerciasis hyper-endemic areas in Sub-Saharan Africa (Aketi, Bas Uélé, Democratic Republic of Congo and potentially an area in Cameroon and the Central African Republic). Participating children will be followed up to determine whether they develop OAE. CSF will be aliquoted and stored at -20°C immediately after collection, with a cold-chain established for transport to the University of Antwerp (UA), Belgium.
Upon arrival, OVRV1 will be detected using qPCR and proteomic strategies. Firstly, qPCR will be performed on cDNA extracted from CSF to determine the presence and quantify OVRV1. Secondly, to identify OVRV1 proteins, reverse-phase liquid chromatography and tandem mass spectrometry (MS/MS) will be performed. The resulting spectra will be processed, and proteins will be identified by comparison with the OVRV1 protein library. The O. volvulus specific O150 qPCR will be performed to detect O. volvulus DNA(9). Additionally, the proteomic datasets will be analysed to search for O. volvulus ESP.

Research question 3
Are OVRV1, O. volvulus mf and/or ESP able to cross the BBB?
Method
Use of an in vitro BBB model
I will use a commercial in vitro BBB model (FUJIFILM Cellular Dynamics). Following establishment of the model, the culture media will be adjusted(10), assessing the influence of permeability on migration of OVRV1 and O. volvulus ESP across the BBB. The transwell inserts will contain brain microvascular endothelial cells (BMECs) on the apical side. To mimic the physiology and functional responses of the BBB inclusion of astrocytes and pericytes is necessary. Specifically, these cells are cultured on the basolateral membrane of the insert, ensuring adequate paracellular tightness and modulate BMEC function(11).
The set-up will be replicated multiple times using different sample types, incl. OVRV1 viral titers and homogenised O. volvulus adult worms. Viral titers will be provided by collaborators at LSTM, where I will learn to maintain the viral culture in Antwerp. Additionally, the infection status of homogenized O. volvulus with OVRV1 will be assessed using the developed qPCR. Furthermore, an initial proteome of the entire worm sample will be established according to the same protocol as previously described. Afterwards, homogenized samples and/or viral titers will be added to the apical medium prior to incubation. Subsequently, the apical medium, the basolateral medium and the BBB cells will be harvested and analysed for OVRV1 and/or O. volvulus DNA/ESP using the methods previously described. If proteins from either OVRV1 or O. volvulus are detected in the basolateral medium, these will be validated as potential biomarkers.