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.

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

Our proposed project focus on basic science of CCHFV. Our identified mutations which affect virus replication and reactivity to the host immune responses may lead significant information on vaccine and antivirals development for CCHFV.

CCHF is a frequent public health threat in endemic countries in Central Asia and the Middle East. Recently CCHF has started to cause public health concerns in South European countries associated with new cases in countries where CCHFV has emerged (which were previously CCHFV free) (1,2). Furthermore, ever-changing environmental disturbances are leading to the expansion of CCHFV regions and the occurrence of large CCHF outbreaks, as seen in India for example (3). Significantly, CCHFV has been listed as a priority pathogen identified by the WHO R&D Blueprint, as requiring new research and development to underpin new interventions. This study will identify patterns of CCHFV genetic variation in humans. Using this data, we will also identify functional mutations which may affect the replication capacity and virulence in humans. Insights of the genetics of CCHFV will lead to a better understanding of how the deadly virus evolves in human hosts, and what selection pressures the virus is under during replication. Furthermore, the identification of mutations which lead to changes in replication dynamics may point to the genetic factors critical for high pathogenicity in humans. Thus, our results will help understand CCHFV evolution and pathogenicity. The successful identification of genetic factors critical for CCHFV pathogenicity in humans, can also be applied to related research on other human pathogenic tick-borne viruses, such as Tick-borne encephalitis virus and Severe fever with thrombocytopenia syndrome virus; two important viruses for the UK and Japan. The data generated in this project will also provide fundamental information about novel targets for antivirals against CCHFV and new vaccine approaches. It will also enable us to monitor the spread of CCHFV strains and their pathogenic risk to humans, in much the same way as fluxes in highly pathogenic avian influenza virus are monitored. Ultimately this work will reduce the threat of CCHF epidemics in humans as well as domestic animals.

Japan Co-PI Kurosaki & UK Co-investigator Hewson are involved in collaborative CCHFV research supported by JSPS. Our proposed project will develop the current CCHFV research partnership between the UK & Japan, enabling a multidisciplinary approach to implement in vitro, in vivo and in silico studies on genetics and virology of CCHFV. We anticipate the outcomes of this work will develop new knowledge and new approaches to control CCHF in the future. Furthermore, the project will enable us to develop our partnership on other tick-borne viruses and haemorrhagic fever viruses. LSHTM & Nagasaki University established a partnership in the research and education for tropical medicine and global health. In 2018, Joint-PhD program between two schools was started. This collaborative project will engage early career researchers and strengthen the relationships in academic research of the schools. Nagasaki University is now constructing BSL4 facility to strengthen the research capacities for highly pathogenic viruses in Japan. The partnership between PHE and Nagasaki University will enhance the opportunities of collaboration between UK and Japan for haemorrhagic fever virus research. LSHTM & PHE have established collaborations, and this new partnership will develop opportunities between these institutes to understand virus pathogenesis and mechanisms of virus emergence. UK Co-PI Allen established a research group at LSHTM, developing extensive collaborations in laboratory sciences with the National Virus Reference Department at PHE. Expanding LSHTM-PHE collaboration to National Virology & Pathogenesis Group with UK Co-Investigator Hewson will create a platform for cutting-edge fundamental and applied research.