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

Crimean-Congo haemorrhagic fever (CCHF) is the most widespread tick-associated viral haemorrhagic disease. Clinical presentation of the disease ranges from non-symptomatic to severe haemorrhage fever leading to a high degree of morbidity and death. The agent of CCHF is the virus CCHF orthonairovirus (CCHFV), a member of the Nairoviridae family with a tripartite negative sense RNA genome. Segment reassortment and recombination between and within segments in addition to genetic drift brought about by the high error rate of the viral polymerase, provides an enormous diversity of genome opportunities, which enables these viruses to adapt, exploit varying conditions and new environmental niches. Within this broad genomic diversity and range in clinical presentation, we expect that genetic determinants of the virus contribute to its pathogenicity in humans. Although no clear evidence is available.

In this research proposal, we aim to determine the virological significance of genetic diversities of CCHFV in human pathogenicity. We hypothesize that CCHFV in humans undergoes distinct genetic variation after introduction from tick or vertebrate hosts, in order to adapt to this new environment, and that the resulting mutations modify viral replication capacities and subsequently virulence in humans.

To this end, the project will focus on the following work packages;

1. Identification of amino acid mutations that have occurred specifically in human isolates.

2. Tracking the emergence of mutations on CCHFV in human cases.

3. Experimental evaluation of the identified mutations.

These results will provide insight into a better understanding of the molecular mechanisms of pathogenicity associated with CCHFV in humans. Additionally, sequence data from various tick samples and human infections will support a better understanding of CCHFV evolution during infection in human and circulation in host ticks. Furthermore, these results will provide information for the potential targeting of vaccines and antivirals against CCHFV.

Technical Summary

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