Molecular Mechanism of IMD (NF-kB) Inhibition by Dengue Virus in the Mosquito Aedes Aegypti, and Implications for Transmission and Emergence

Dengue virus is the most important mosquito-borne virus causing human disease. Almost half the world's population is at risk of dengue virus infection, mostly in tropical low and middle-income countries. Approximately 400 million people are infected each year, with about 100 million cases of the severe flu-like dengue fever and 500,000 cases of the more severe and potentially fatal dengue haemorrhagic fever. The major impact of dengue disease, on top of significant suffering and loss of life and economic productivity, is that underdeveloped healthcare systems are overwhelmed during the almost annual epidemics experienced by many countries, affecting routine and emergency medical care. There are no medicines to treat dengue and the only licensed vaccine is imperfect and not recommended for widespread global use.

Dengue virus is transmitted by mosquitoes and cannot spread directly between humans. When a mosquito feeds on an infected person, it too becomes infected with dengue virus. The virus is passed on when the mosquito feeds on another person, causing that second person to become infected. Preventing transmission through the mosquito is therefore an effective way of reducing the global disease burden.

The mosquito that usually transmits dengue virus is the 'yellow fever mosquito' (Latin name Aedes aegypti). Like humans, mosquitoes have an immune system that protects them against viral diseases. The immune response of the yellow fever mosquito springs into action when the insect is infected with viruses, but not all arms of the immune system are able to fight dengue virus. We discovered that this is because dengue virus actively blocks certain parts of the immune response. This grant will investigate how dengue virus escapes from these arms of the immune system, a first for any mosquito-borne virus. Ultimately, this will allow us to develop ways of strengthening the immune system of mosquitoes to stop them from being infected with dengue virus, which will reduce transmission and protect people from dengue disease.

Our first goal is to work out how dengue virus blocks mosquito immune responses. Dengue virus makes ten proteins when it infects a mosquito, and we predict that one of these proteins disrupts mosquito immune responses. We will identify which protein can do this, and how. Our second goal will look at this question from a different angle by asking how important certain arms of the mosquito's immune response are for fighting dengue infection. We will do this by looking at what happens to the virus when we grow it in mosquito cells that lack these parts of the immune response. These first two goals will be researched using cells taken from mosquitoes, which are easy to work with in the lab and useful for finding out the fine details of how dengue virus hides from the mosquito immune system. However, we cannot use these cells to study transmission. For this reason, the third and final goal of this grant is to repeat key experiments from the first two goals in yellow fever mosquitoes in the laboratory to check that our results are relevant to what happens in an actual mosquito. There are four different strains of dengue virus, some of which behave very differently from one another, so we will also check whether our results can be extrapolated to all four dengue strains.

Together, our experiments will tell us how dengue virus blocks mosquito immune responses, and how important the ability to escape certain mosquito immune responses is for the transmission of dengue virus. In the future, this information will allow us to reduce the global burden of dengue disease by making the yellow fever mosquito's immune response stronger and better able to fight dengue virus in order to prevent disease transmission.

Dengue virus (DENV) is the most important mosquito-borne virus of humans, with almost half the world's population at risk in (mostly) low and middle-income countries across the tropics. There are 390 million infections annually, with 96 million cases of dengue fever and 500,000 cases of the potentially fatal dengue haemorrhagic fever. No antivirals are available, and the only licensed vaccine has mixed efficacy. Mosquito control is therefore important for reducing the global disease burden. Vector immunity restricts DENV replication and transmission. However, we showed that DENV-2 inhibits the IMD pathway, an NF-kB response analogous to human TNF receptor signalling, in cell lines derived from Aedes aegypti, the major DENV vector. This grant will (i) characterise the molecular mechanism of DENV-2's IMD antagonism; (ii) evaluate the impact IMD responses have on DENV-2 evolution; (iii) establish the relevance to diverse DENV serotypes and transmission potential in vivo. Aim 1 exploits our unique IMD assays and track record in Ae. aegypti proteomics to identify which DENV-2 protein antagonises IMD activation, along with its cellular targets. In aim 2, which is not dependent on aim 1, we will experimentally evolve DENV-2 in our first-in-field IMD-deficient CRISPR cell lines to assess the impact on the viral quasispecies (the total population of viral sequences) and identify viral sequences required for IMD antagonism. Results will be validated in an established reverse genetics system. Aim 3 will establish real-world relevance by testing transmission potential in mosquitoes infected with wild-type DENV-2 and IMD-sensitive mutants from aim 2. Key experiments will also be repeated with divergent DENV serotypes. We will thus define virus-vector interactions modulating DENV transmission, with implications for ongoing epidemics and future opportunities for vector-targeted control strategies. This will be the first characterisation of mosquito NF-kB evasion for any arbovirus.

Our research will improve our understanding of mosquito-borne virus transmission and emergence, with positive impacts in the UK and internationally. Our work aligns well with the UK's commitment to tropical disease research through the £1.5 billion Global Challenges Research Fund, part of the country's Official Development Assistance commitment.


HEALTH AND WELLBEING

Dengue predominantly affects the urban poor in low- and middle-income countries across the tropics. The future development of our findings into mosquitoes less able to transmit dengue virus and other viruses would have a huge global impact. Morbidity and mortality would be reduced for the 2.5 billion people at risk of dengue disease, resulting in increased school attendance, reduced working days lost, improved healthcare (severely stretched during dengue epidemics) and improved economic opportunity. Dengue virus is not endemic in the UK, although imported cases are reported each year. Our greatest national impact will be through our characterisation of broadly applicable principles underpinning the transmission of local vector-borne viruses of livestock (e.g. bluetongue virus). Reducing transmission of these diseases would increase agricultural productivity and food security locally and internationally.

Public interest in emerging diseases, especially the mosquito-borne Zika virus (which is closely related to dengue virus), is high. Our work will identify adaptations linked to the emergence of mosquito-borne diseases. Although predicting viral emergence is difficult, our work will contribute tangibly to discussions about real and perceived risks from these diseases at home and abroad.


COMMERCIAL PRIVATE SECTOR

The UK is a global leader in vector-borne disease research, with one of the world's leading companies in genetically modified mosquitoes (Oxitec) located here. Our research will identify new ways of modifying mosquitoes to prevent the transmission of vector-borne diseases afflicting humans and livestock, which could contribute to new products for the global market. Our findings will be transferrable to applications making livestock species (e.g. honeybees, silkworms) more disease resistant, and for the biological control of agricultural pest insects. Thus, our research could stimulate private sector growth into new markets. This would create jobs and would strengthen the UK's competitive advantage in this sector.


ECONOMIC COMPETITIVENESS

The UK's competitiveness depends on the economic health of our trade partners. The global annual economic cost of dengue is upward of US$9 billion. With dengue endemic in 41 of the 52 Commonwealth nations (including India), with which we have strong ties, and the US and China also at risk, our work has a potentially large long-term economic impact if our findings help reduce the burden of dengue disease. If our work leads to applications that improve agricultural productivity, we will also impact this UK industry (7% of total UK economy).


POLICY DEVELOPMENT

The recent emergence of Ebola and Zika virus has thrown emerging diseases into the spotlight, with several parliamentary sessions dedicated to this topic. Our research is relevant to policies aimed at protecting the UK population from emerging viruses, and international policies relevant to dengue endemic partner countries.


PROVISION OF SKILLED PEOPLE IN THE WORKFORCE

Vector-borne diseases are predicted to cause an increasing disease burden in the UK and globally as climate warms and vectors encroach on new areas. The scientist on this grant will be trained in a highly specialised scientific skillset and broadly transferrable skills in oral and written communication, public engagement, multi-centre collaboration and student supervision. This will prepare them for a career as a future scientific leader, or in teaching, policy or public engagement, which are ever-more important as vector-borne diseases increase in global importance.