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

Lead Research Organisation: University of Surrey
Department Name: Microbial & Cellular Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description International Society for Neglected Tropical Diseases Dengue Advisory Group
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
URL https://www.isntd.org/dengue-advisory-group
 
Description MSc research training
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact The immediate outcome of this impact activity is that these Masters students went on to the following subsequent career destinations: PhD programme: 2 students Technical position in research institution: 1 student
 
Description Medical Microbiology MSc (University of Surrey)
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact This course trained approximately 10 students each year to be specialists in medical microbiology. A number of these students have gone on to do PhDs in leading institutions (for example The Pirbright Institute) in this field. Others continue to work at UKPHA, APHA and other governmental organisations in the UK and abroad.
 
Description Microbiology Society Members Panel
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
URL https://microbiologysociety.org/why-microbiology-matters/council-governance/standing-panels/members-...
 
Description Microbiology Society Policy Committee
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
 
Description PhD research training
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact The concrete outcomes of this PhD training was the skills learnt by the respective students while under my guidance. Their immediate destinations after graduating were: Postdoctoral academic research (US): one student Postdoctoral academic research (UK): one student
 
Description Veterinary Microbiology MSc course (University of Surrey)
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact This course trained approximately 10 students each year to be specialists in veterinary microbiology. A number of these students have gone on to do PhDs in leading institutions (for example The Pirbright Institute) in this field. Others continue to work at UKPHA, APHA and other governmental organisations in the UK and abroad.
 
Description A One Health Approach to Alkhumra Haemorrhagic Fever Virus Species Tropism, Pathogenesis and Epidemiology
Amount £99,082 (GBP)
Organisation The Pirbright Institute 
Sector Academic/University
Country United Kingdom
Start 09/2022 
End 04/2026
 
Description A new paradigm for arbovirus emergence: how transposons modulate mosquito vector competence
Amount £19,701 (GBP)
Funding ID RGS\R1\191486 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2019 
End 06/2020
 
Description Defining the molecular signature of pericyte dysfunction in severe dengue patients
Amount $20,000 (USD)
Organisation UGPN 
Sector Academic/University
Start 09/2020 
End 07/2021
 
Description Dengue haemorrhage: a novel role for perivascular cells
Amount £74,137 (GBP)
Organisation University of Surrey 
Sector Academic/University
Country United Kingdom
Start 09/2021 
End 09/2024
 
Description Floid Cell imager (X2)
Amount £24,459 (GBP)
Organisation The Pirbright Institute 
Sector Academic/University
Country United Kingdom
Start 11/2021 
End 03/2022
 
Description Global characterisation of interlinked mosquito immune networks
Amount £9,000 (GBP)
Organisation The Pirbright Institute 
Sector Academic/University
Country United Kingdom
Start 07/2021 
End 03/2022
 
Description Harry Smith Vacation Studentship
Amount £2,320 (GBP)
Funding ID GA002363 
Organisation Microbiology Society 
Sector Learned Society
Country United Kingdom
Start 06/2020 
End 08/2020
 
Description Identification of protein sensor of viral RNA in mosquito Aedes aegypti IMD pathways
Amount £79,703 (GBP)
Organisation University of Surrey 
Sector Academic/University
Country United Kingdom
Start 09/2019 
End 08/2023
 
Description The battle for the 5' end: dissecting a novel virus-specific translation mechanism driven by eIF3
Amount £402,936 (GBP)
Funding ID BB/S006931/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2019 
End 01/2023
 
Title CRISPR knockouts of RNAi pathway components in Aedes aegypti Aag2 cells 
Description We developed a number of CRISPR knockout cell lines based on the Aedes aegypti Aag2 cell line. These cell lines have facilitated research on virus-host interactions in the mosquito vector, and are also facilitating studies into the fundamental nature of antiviral responses of mosquitoes. 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? Yes  
Impact We have distributed these cell lines to numerous research groups across the world, including in the US, many European countries and research groups across the UK. These groups have made significant discoveries using these tools, many of which are published. Some of these cell lines were also made available via ECACC (https://www.culturecollections.org.uk/collections/ecacc.aspx). 
 
Title Clonal Aedes aegypti mosquito cell lines 
Description We took an existing Aedes aegypti mosquito cell line (Aag2) as a starting point, and generated clonal-derived cell lines that are better defined on a number of parameters and that have in some cases been 'cured' of persistent viruses contaminating the original cell line. 
Type Of Material Cell line 
Year Produced 2019 
Provided To Others? Yes  
Impact The cell lines have so far been distributed to dozens of research groups across the world, including in the USA, Netherlands, Germany, France, India and UK (among others). Certain cell lines were also deposited with ECACC (phe-culturecollections.org.uk) and these are now available internationally through this public repository. These cell lines have significantly improved reproducibility and standardisation across the field, as they have provided a single, well-characterised source of cells that different research groups can access for which the history is well documented. 
URL https://www.phe-culturecollections.org.uk/products/celllines/generalcell/search.jsp?searchtext=aag2&...
 
Title Fluorescence protein expression plasmids 
Description We developed plasmids for expressing fluorescence proteins (e.g. mCherry) in mosquito cells, as an experimental control or for measuring protein localisation and transfection efficiency. The plasmids were deposited with Addgene with reference number 154164. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact These plasmids have been requested 10 times by researchers in USA, Netherlands, Austria, Japan. 
URL https://www.addgene.org/
 
Title Luciferase reporter plasmids for detecting mosquito NF-kB immune pathway activation 
Description We developed the first luciferase-based reporter plasmids that are based on Aedes aegypti mosquito NF-kB response elements for detecting the activation of mosquito NF-kB pathways. These luciferase assays have been deposited with Addgene.org with reference numbers 154152-154163. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact These resources have been requested 1 time by research groups in China. 
URL https://www.addgene.org/
 
Title Luciferase reporter plasmids for measuring insect RNA interference activity 
Description We developed and validated luciferase reporter assays that can be used to measure insect immune responses - specifically the RNA interference pathway. These plasmids were deposited with Addgene with reference numbers 123655 and 123656. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact The plamids have been accessed 13 times by researchers in Australia, Singapore, USA, Germany, Czechia, India. 
URL https://www.addgene.org/
 
Title Plasmids expressing Aedes aegypti NF-kB transcription factors 
Description These plasmids express Aedes aegypti transcription factors related to mosquito NF-kB immune pathways. The plasmids are useful for studying immune responses in mosquitoes and have been made availabe via Addgene with reference numbers 154165-154168. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact Unknown so far. 
URL https://www.addgene.org/
 
Description Dengue microvascular pathogenesis 
Organisation University of Surrey
Department Faculty of Health and Medical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I brought my expertise in dengue and virology, and had significant intellectual input into shaping the research question and methodological approach. I also directly contributed to the training of one shared MSc student, one shared PhD student and three undergraduate students who have contributed to this project so far.
Collaborator Contribution The partner (Dr. Paola Campagnolo, University of Surrey) brought their expertise in cardiovascular biology and had significant intellectual input into shaping the research question and methodological approach. They also directly contributed to the training of one shared MSc student, one shared PhD student and three undergraduate students who have contributed to this project so far.
Impact The collaboration has resulted in the following publications: DOI: 10.1128/mSphere.00258-20 DOI: 10.1128/mSphere.00258-20 This is an interdisciplinary collaboration. The disciplines are microbiology/virology (me) and cardiovascular biology (Dr. Paola Campagnolo, University of Surrey).
Start Year 2018
 
Description Exploiting mosquito immunity for generating refractory mosquitoes 
Organisation The Pirbright Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution I brought my expertise in mosquito immunology and viral evasion of innate immune responses in vertebrates and invertebrates. I provided intellectual input into the project and provided training for one research assistant.
Collaborator Contribution The partner (Luke Alphey, The Pirbright Institute) brought their expertise in mosquito transgenesis and molecular approaches to developing refractory mosquitoes that cannot transmit viruses. The partner also provided intellectual input into the project.
Impact The following publications are associated with this collaboration: DOI: 10.3390/v13112116
Start Year 2020
 
Description Flavivirus-peroxisome interactions 
Organisation University of Surrey
Department Faculty of Health and Medical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I contributed my expertise in molecular virology of flaviviruses, and my expertise in high containment research for working with ACDP Hazard Group 3 and Schedule 5 viruses. I provided access and performed experiments in the required high containment facilities. I provided intellectual input into the project and supported the training of one joint PhD student.
Collaborator Contribution The partner (Dr. Peter Mayerhofer, University of Surrey) contributed their expertise in peroxisome cell biology and biochemistry. They also provided intellectual input into the collaboration and provided training to the joint PhD student.
Impact The following publications resulted from this partnership: DOI: 10.3390/v14020253
Start Year 2019
 
Description Role of Ago2 in mosquito antiviral RNAi 
Organisation Bernhard Nocht Institute for Tropical Medicine
Country Germany 
Sector Academic/University 
PI Contribution I provided my expertise in generating CRISPR knockout cell lines in mosquito cells and shared these reagents with the collaborators.
Collaborator Contribution The collaborators brought their expertise in RNAi antiviral immunity in mosquitoes. They also brought their intellectual input and trained their own staff on the project.
Impact The following publications are associated with this research: DOI: 10.3390/v13061066
Start Year 2018
 
Description Role of Ago2 in mosquito antiviral RNAi 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution I provided my expertise in generating CRISPR knockout cell lines in mosquito cells and shared these reagents with the collaborators.
Collaborator Contribution The collaborators brought their expertise in RNAi antiviral immunity in mosquitoes. They also brought their intellectual input and trained their own staff on the project.
Impact The following publications are associated with this research: DOI: 10.3390/v13061066
Start Year 2018
 
Description Expert commentary on New Scientist article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I provided expert commentary on an article published in New Scientist, which summarised research performed by researchers at the University of Leeds on how mosquito bites enhance the transmission of vector-borne diseases.
Year(s) Of Engagement Activity 2020
URL https://institutions.newscientist.com/article/2231141-skin-cream-applied-to-mosquito-bites-stops-vir...
 
Description MRC PhD Student DTP 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I gave a career development seminar to the MRC PhD student DTP to discuss opportunities and realities of an academic research career in universities and research institutions.
Year(s) Of Engagement Activity 2021
 
Description Microbiology Today Article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I authored a lay article on dengue virus and current research topics in the lab and the wider field. The purpose was to generally inform the public, students and professionals on the major aspects of dengue disease as part of a wider magazine issue (print and online) focussed on arthropod-borne viruses.
Year(s) Of Engagement Activity 2019
URL https://microbiologysociety.org/publication/past-issues/arboviruses-and-their-vectors/article/dengue...
 
Description Microbiology practical: engagement for underrepresented school children 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This was a public outreach activity to introduce school children from underrepresented communities to the concept of attending university, and topics and practical classes related to a degree in microbiology.
Year(s) Of Engagement Activity 2018,2019
 
Description World Dengue Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was the inaugural World Dengue Day, an annual multi-day series of events to raise awareness of the global impact of dengue. The event raised awareness of dengue globally and sparked interest from a number of attendees in our research.
Year(s) Of Engagement Activity 2021
URL https://www.adva.asia/world-dengue-day-2021/