Analysis of flavivirus infection on the cellular lipidome - implications for virus particle production and replication.

Flaviviruses are arthropod-borne RNA viruses that cause significant human morbidity and mortality worldwide. Over the last 50 years, a number of flaviviruses including; dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV) and recently Zika virus (ZIKV) have emerged to cause diseases that are now serious global public health concerns. Countries in tropical and sub-tropical regions, such as Thailand, bear the greatest economic and societal cost of these diseases, which are endemic due to the presence of the mosquito vectors. It is estimated that DENV infections alone cost Thailand some US$290 million per year in direct and indirect costs. However, fully protective and safe vaccines for prevention of mosquito-borne flavivirus diseases are only available for yellow fever virus (YFV) and JEV. As such, there is a need to develop improved flavivirus vaccines and anti-viral therapies.

A greater understanding of the interaction between flaviviruses and their hosts will facilitate improved control measures. Previous studies have established that flaviviruses modulate cellular lipid metabolism to promote virus replication and assembly and that changes in serum lipids are associated with disease severity. However, there is little known concerning how the overall changes to lipid metabolism during infection specifically affect i) the lipid composition of the virus particle and its infectivity ii) the types of lipids that are secreted from infected cells and iii) disease processes. Little is also known concerning how different flaviviruses alter the host and virus lipidome and whether common lipids are involved.

The UK and Thai investigators have already conducted preliminary studies using high-throughput mass spectrometry (MS) to investigate how DENV infection i) modulates intracellular and secreted protein profiles ii) modulates the cellular lipidome and iii) influences the composition of DENV particles. The studies have revealed that proteins involved in lipid metabolism and/or lipoproteins are differentially regulated and secreted during DENV infection and that the lipidomic profile of DENV-2 infected cells is altered, compared to uninfected cells.

In this investigation we propose to use advanced high-throughput liquid chromatography-MS to analyse how lipid metabolism is perturbed during flavivirus infection and specifically define the lipids that are key for flavivirus replication and particle assembly. Specifically, we will examine the lipid content of infectious flavivirus particles and virus-like-particles (VLPs) and the cellular membranes used for flavivirus replication during infection. We will also determine whether flavivirus infection results in changes in the lipids secreted from cells. The studies will be done using three flaviviruses of concern in Thailand; DENV, JEV and ZIKV, to determine if flaviviruses rely on common changes to lipid metabolism for their replication and particle assembly. The importance of these lipids in infection will be confirmed by altering their synthesis by RNA knockdown and/or drug treatment. We will also determine whether the lipid composition of virus particles affects the host response to viral entry and the immunogenicity of virus particles.

In the longer term, this knowledge will be used as a basis to i) manipulate cellular lipid synthesis to increase the secretion of VLPs to produce improved vaccines ii) identify lipid biosynthetic pathways essential for flavivirus replication and potentially targets for novel anti-viral therapies and iii) increase our knowledge of viral pathogenesis. The project will build on existing collaborations between the UK and Thai partners and develop the capability of the Thai partners to use high-throughput approaches to analyse virus particles and how viral infection remodels the cellular lipidome, not only for flaviviruses, but also for other emerging arboviruses for which no therapies exist.

The overall aim of the UK-Thai collaboration is to increase our understanding of how flavivirus infection impacts cellular lipid metabolism and identify cellular lipids that are key for flavivirus replication and particle assembly.

Initially human hepatic Huh-7 cells will be infected with the four serotypes of DENV, JEV and ZIKV, which are flaviviruses of most relevance to Thailand. The infected and mock infected cells will be used to prepare total cell lysates and a heavy membrane fraction containing the virus replication/assembly complex. We will also collect the culture supernatants from the virus infected cells and use them to isolate virus particles, a total supernatant fraction and exosomes.

Lipids extracted from the samples will be analysed by ultraperformance liquid chromatography - mass spectrometry (UPLC-MS). The large datasets resulting from the analyses will be subject to bioinformatic analysis to identify classes of lipids modulated during virus infection. More detailed targeted analysis will be undertaken to identify lipid species that are either associated with flavivirus replication and/or particle morphogenesis or modulated in response to infection with multiple flaviviruses.
The role of the identified lipids in the flavivirus lifecycle will be assessed by i) localisation of the lipids by immunological techniques and ii) siRNA knockdown or compound inhibition of key proteins involved in lipid metabolism, in combination with flavivirus replicon and infection assays. The findings will be validated using a monocyte-derived dendritic cell infection model.

The lipid composition of recombinant virus-like-particles (VLP) will be analysed to determine if it differs from virus particles and whether the composition depends on remodelling of host membranes during virus replication. The effects of differences in VLP lipid composition will be analysed by examining the effects on VLP yield, immunogenicity and host cell signalling.

The project will have immediate, medium and long-term benefits.

The project involves the use of cutting edge lipidomics and broad bioinformatics analysis skills. At the beginning of the project it is planned to hold a workshop in Thailand to train the Thai investigators in advanced mass spectrometry based lipidomic and proteomics analysis. The workshop will be open to Thai scientists in general, not only those affiliated with the project. The UK PI recently organised a UK-Thai workshop "Bristol Tackles Global Challenges" (Bristol, March, 2017 - 24 Thai scientists) to promote collaborative research between UK and Thai scientists, and will build on the networks made, to ensure the proposed workshop has maximum impact for Thai scientists.

In the medium term, as the project progresses and large lipidomic datasets are produced, the UoB and particularly the Thai PDRAs will acquire advanced training in lipidomic bioinformatic analysis skills that can be applied to other research investigations outside the field. In addition to the staff employed directly under the grant, the project will serve as a training platform for a number of Thai students, at both M.Sc. and Ph.D. level. These students will benefit from the advanced methodological and bioinformatics training, as well as benefit directly by being exposed to a wider international research environment. Two Thai students (1 M.Sc. and 1 PhD) from the Thai PIs lab have already undertaken 3 month research attachments in the UoB PI's lab, and it is planned that this will be significantly expanded as a consequence of this research program. In addition, the UK PI currently supervises a Thai PhD student (supported by the Faculty of Tropical Medicine, MU) who is being trained in advanced proteomic analysis and will also be involved in the proposed programme and able to transfer her skills in proteomics and lipidomics to Thai researchers in the future.

The long-term beneficiaries will be the citizens of countries where flaviviral diseases are endemic, many of who are in middle and lower income countries, including Thailand. While this application focuses on mosquito-borne flaviviruses, the scientific approach developed in this proposal can also be adopted against other emerging viral diseases that disproportionally affect developing countries such as Thailand. The approach taken in this proposal is intensively data driven, relying on data produced in the project, but also publicly available data. This means that once suitably trained, researchers in countries like Thailand will have the ability to "mine" publicly available datasets to identify targets and compounds that can be tested for activity against emerging viruses. As these compounds may already be licensed there is the potential for a researcher in a resource poorer setting to find more cost effective solutions to combat emerging viral infections, which will have direct benefits to the health and wealth of the nation.

Outside of academic beneficiaries, the major immediate beneficiaries of the results produced in the investigation will be those in the commercial private sector. There is an active programme worldwide to discover vaccines and antiviral therapies against flaviviruses and the knowledge to produce improved vaccines and identify druggable targets will also be of significant interest to those in these fields. As such, the PIs will draw on the commercial contacts developed over many years of active research against flaviviruses (with dengue the most advanced) to maximise the impact of the research findings (as described in the Pathways to Impact statement). Therefore, the PIs have a number of options that can be pursued to translate the basic research findings from the investigation into more developed antiviral strategies.