Title of PhD project
Molecular biology of Crimean-Congo haemorrhagic fever virus: Diagnostics, reverse genetics and vaccine studies
Lead: Roger Hewson (Faculty of Infectious and Tropical Diseases, firstname.lastname@example.org)
Professor Hewson is a recently appointed professor at LSHTM, where he specialises in molecular virology of arboviruses and viral haemorrhagic fevers. He also maintains a part-time role in the UK-Health Security Agency (UKHSA) at Porton Down where he is involved in BSL4 work on a number or emerging pathogens. He has worked on Crimean-Congo haemorrhagic fever virus (CCHFV) for over 20 years, building and developing a wide range of international collaborations in endemic countries. Hewson -san will be the lead supervisor.
Professor Sakurai (email@example.com).
Professor Sakurai is a member of NU School of Tropical Medicine and Global Health (TMGH) where he will become a professor of the PhD course at TMGH. He is also a member of the Institute of Tropical Medicine where he is a member of the Emerging Infectious Disease Department. Sakurai - san will be the secondary supervisor. He has expertise in antivirals, molecular virology and bioinformatics .
Professor Jiro Yasuda (firstname.lastname@example.org) runs the Emerging and Infectious Disease Department at NEKKEN. Yasuda - san will make important contributions to the supervisory team by contributing his specialist knowledge of emerging viruses and their ability to enter, replicate and produce new progeny viruses.
Brief description of project
Provide a brief background and a brief outline the proposed project or theme (including very briefly types of data/study design). If part of a larger study, briefly outline the main study and the additional importance of this proposed work. (Up to 5 relevant references are allowed):
The Nairoviridae family within the Bunyavirales order comprises tick-borne, tri-segmented, negative strand RNA viruses, with several members being associated with serious diseases in humans and animals. A notable member is Crimean-Congo haemorrhagic fever virus (CCHFV) - the most widely distributed tick-borne pathogen associated with severe human disease. Case fatality rates of CCHF average 30% but can reach 80% when associated with direct human – human transmission. CCHFV is a global threat sensitive to climate change, it has recently emerged in Spain and is highlighted by the WHO R&D blueprint as an emerging pathogen requiring urgent further research1. Its highly pathogenic nature combined with the lack of any licenced vaccines or therapeutics mandates all work with infectious virus is conducted in BSL4 facilities, such as those at the UKHSA institute at Porton Down (UK) and the BSL4 facility soon to be opened at Nagasaki University.
In this work the student will use molecular biology approaches for project activity on new diagnostics and the development of novel research tools for CCHFV. It is envisaged that the student will use these developments for vaccine research and to study the pathogenicity of different CCHFV strains.
New CCHFV diagnostics will be focused on simple assays that can be used in low resource rural settings, where CCHFV transmission typically occurs. The Yasuda & Hewson labs have specialised in the development rapid diagnostics for several other viral haemorrhagic fevers and the student will benefit from this background1,2. In previous work Hewson has developed a reverse genetics methodology for the closely related Nairoviridae family member Hazara virus3. The student will use this background to identify any opportunities which may arise to assess antivirals; but also to develop a related methodology for CCHFV and incorporate a strategy to spit the CCHFV genome such that non-infectious CCHFV can be produced in low (BSL2) containment, (an approach used previously for Lassa fever virus4). This non-infectious material may be suitable for new CCHFV vaccine approaches. Which the student will investigate. The developed reverse genetics capability for CCHFV will be used to study the genetic contribution different virus sequences have on replication and pathogenicity. Hewson is involved in a long-term collaboration with colleagues in Turkey who have collected and sequenced CCHFV from mild and sever cases of CCHF disease, these sequences and subsequently recombinant viruses (made in this project) will be studied in vitro at Hewson BSL4 laboratory in Porton Down. Over the course of this work the student will also transition CCHFV virology to the new BSL4 facility in Nagasaki – building on our future and long term collaborative plans between these world leading institution in support of emerging viruses of pandemic potential.
Pemba CM, Kurosaki Y, Yoshikawa R, Oloniniyi OK, Urata S, Sueyoshi M, Zadeh VR, Nwafor I, Iroezindu MO, Ajayi NA, Chukwubike CM, Chika-Igwenyi NM, Ndu AC, Nwidi DU, Maehira Y, Unigwe US, Ojide CK, Onwe EO, Yasuda J. Development of an RT-LAMP assay for the detection of Lassa viruses in southeast and south-central Nigeria. J Virol Methods. 2019 Jul;269:30-37. doi: 10.1016/j.jviromet.2019.04.010.
Bonney LC, Watson RJ, Afrough B, Mullojonova M, Dzhuraeva V, Tishkova F, Hewson R. A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection. PLoS Negl Trop Dis. 2017 Oct 13;11(10):e0006013. doi: 10.1371/journal.
Fuller J, Surtees RA, Slack GS, Mankouri J, Hewson R, Barr JN. Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage. J Virol. 2019 Jul 17;93(15):e00616-19. doi: 10.1128/JVI.00616-19.
Kainulainen MH, Spengler JR, Welch SR, Coleman-McCray JD, Harmon JR, Scholte FEM, Goldsmith CS, Nichol ST, Albariño CG, Spiropoulou CF. Protection From Lethal Lassa Disease Can Be Achieved Both Before and After Virus Exposure by Administration of Single-Cycle Replicating Lassa Virus Replicon Particles. J Infect Dis. 2019 Sep 13;220(8):1281-1289. doi: 10.1093/infdis/jiz284.
The role of LSHTM and NU in this collaborative project
A range of CCHF viruses are available in Hewson’s BSL4 laboratory (including RNA at LSHTM), these have provided high quality full-length genomes which in this project, will be shared with the supervisory team at Yasuda lab at NU. Working closely with the team the student initially based at LSHTM will compare sequences with data available in electronic databases. Using previous expertise provided by NU and LSHTM (inc. UKHSA) an isothermal assay will be designed. This may involve student visiting NU, detailed plans will be developed in due course. At LSHTM the student will also develop cell culture capability at BSL2 and work up skills with Hazara virus (HAZV), this will include learning and using the HAZV reverse genetics system. The Sakurai and Yasuda team at NU will contribute their expertise on viral replication and if necessary, suggest novel approaches to study antivirals. Sakurai is Assistant Professor in the Yasuda lab (Emerging Infectious Disease) at the Institute of Tropical Medicine at NU (NEKKEN). Rescued HAZV which will be made by the student, will have a Green Fluorescent Protein (GFP) reporter tag which could support high throughput antiviral screening campaigns in which Sakurai at NU has particular expertise. Based on the highly efficient HAZV reverse genetics system the student will develop clones and plasmids using CCHFV sequences. A GFP reporter virus will be developed and initially rescued in the UK BSL4 laboratory at Porton Down (linked to Hewson’s lab at LSHTM), wild type virus will also be rescued. Cloning and plasmid construction work will be supported by Hewson and the Yasuda lab through regular lab meetings including over zoom. This platform will also be amenable to the design of a non-infectious virus like particle (VLP) system supported by joint expertise at NU and LSHTM. Upon completion of these steps the student will investigate VLP vaccine opportunities by assessing the immunogenicity of non-infectious VLPs with various adjuvant systems in experimental animal models. In addition to the supervisory team’s wherewithal in vaccinology, the student will also benefit from expertise provided by the Vaccine Centre at LSHTM. Following the demonstration of induced immunity, vaccine efficacy experiments will be performed using CCHFV challenge studies which will be undertaken in the BSL4 lab at Porton. The student, nevertheless, will work in close cooperation with the supervisory team and key input from NU and LSHTM will drive the PhD forward. Over the course of this project the student will catalyse linkages between NU, NEKKEN, LSHTM and the UK-HAS BSL4 laboratory.
Particular prior educational requirements for a student undertaking this project
Good first (BSc) degree in biology, MSc or equivalent in Medical Microbiology / Virology. Practical laboratory experience in molecular biology / virology.
Skills we expect a student to develop/acquire whilst pursuing this project
Bioinfomatics skills with a range of computer programmes to compare and contrast viral sequences. The student will develop a working capability with viral RNA and cDNA clones, they will use and work up expertise in polymerase chain reactions and the use of appropriate (rt-PCR) hardware and software to develop and analyse diagnostics and viral load. A developing capability in isothermal diagnostic assays and the readouts of such assays paper media will also develop. Molecular cloning skills will be developed including in the design and construction of appropriate transcription and expression vectors. Work with live Hazara virus at BSL2 gradually working up to BSL4 with CCHFV over the course of the PhD programme is also expected. Virus amplification in vitro, plaque assay at BSL2 working up to BSL3 and BSL4. Cell culture and transfection techniques with mammalian cells at BSL2, construction of recombinant viruses and recombinant non-infectious virus like particles. Vaccine and adjuvant studies in animal models, assessment of immune reactivity of VLP vaccines and challenge studies at BSL4 in vivo with appropriate (muse) models of CCHF disease.