A cross-sectional approach to identification & interrogation of adaptive & functional mutations affecting CCHFV replication & pathogenicity in humans

Identification of CCHFV amino acid mutations: determine unique genetic variation in human, tick & other mammal-isolated CCHFV genome sequences -Analysis of publicly available sequences from in NCBI/EMBL/DDBJ database, identify genetic variation associated with host via bioinformatics technique and virus evolutionary analysis -Map mutations onto known structures of CCHFV proteins to predict structural and biophysical impact of amino acid substitutions on function of viral proteins by computational analysis Tracking the emergence of mutations in CCHFV human cases: examine sequence variants of CCHFV isolates from patients: -CCHFV sequencing in human patients & ticks collected in Turkey using a novel sequencing method (Oxford Nanopore MinION) -Examine genetic variations of CCHFV for intra-host single nucleotide variants (iSNVs) -Combining genetic data of CCHFV isolates with clinical outcomes of cases (fatal vs recovered), highlight genetic determinants of viral pathogenicity Experimental evaluation of identified mutations: Examine functional changes of mutations found in in silico and in vivo work -Create a library of plasmids expressing CCHFV proteins with particular single/multiple amino acid mutations predicted to induce functional changes -Establish viral entry assay using infectious particles/psuedotyped VSV bearing CCHFV envelope in a CCHFV-susceptible human cell line, evaluate infectivity via viral glycoprotein Gn/Gc with host-specific mutations & neutralizing antibody escape mutations -Develop Hazara virus infectious virus particle system into a CCHFV infectious clone to evaluate virus entry & neutralizing antibody escape mutations -Establish CCHFV minigenome in a human cell line to evaluate virus genome replication and transcription -Examine virus replication kinetics in human, non-human primate, and tick cell lines, determine virus sequences of each passage by NGS to examine emergence of genetic variations depending on the host cell species