Tom
van der Most

Project

Track: 14/24, Virome & Immunity in Culex pipiens

Promotor

prof.dr. Ronald van Rij

Co-promoter(s)

dr. Pascal Miesen

Co-supervisors

dr.ir. Gorben Pijlman, dr. Sander Koenraadt

Institute(s)

Radboud Institute for Molecular Life Sciences

Description PhD project

The Virome & Immunity in Culex pipiens PhD project fulfills three goals within the consortium:

Analysis of Dutch field-caught Culex pipiens mosquitoes to determine the presence of arboviruses deemed relevant for the One-Health pact

Presently, we are interested to know how prevalent WNV and USUV are in the Netherlands, sampling both birds and mosquitoes in the same location in order to relate the data to each other. The mosquito species will be determined in Wageningen and samples will be analysed for WNV and USUV presence for this project in Nijmegen (Objective 1).

Increase our understanding of factors influencing vector competence

Arboviruses have to replicate sufficiently in the mosquito host in order to be transmissible through a mosquito bite. The nature of the interaction of the virus with the anti-viral immune response impacts whether the virus is tolerated in the mosquito (and efficiently replicates) or whether the virus is cleared. Vector competence is therefore strongly affected by the anti-viral immune response, although little is known about this response in Culex mosquito species. Apart from arboviruses, mosquitoes are infected with insect-specific viruses, forming the mosquito virome. Similar to arboviruses, these insect-specific viruses interact with the immune response and their presence is thought to affect vector competence of arboviruses. Within this project we therefore aim to increase the understanding of the Culex pipiens immune response (Objective 3) and characterize the virome (Objective 2).

3) Potentially employ natural Culex pipiens viruses as an intervention mechanism restricting arboviral transmission.

In the mosquito species Aedes Aegypti, the presence of the Wolbachia bacterium restricts the vector competence of the mosquito for DENV. Similarly, it is possible that insect-specific viruses in the virome of Culex pipiens mosquitoes restrict vector competence to relevant arboviruses. Insect-specific viruses in the Dutch Culex pipiens virome will therefore be isolated and tested for the restriction of arboviral vector competence (Objective 2b).

Research questions / objectives

Determine prevalence of WNV & USUV in Dutch Culex pipiens
Characterize the natural virome of Dutch Culex pipiens
1. Correlate individual viral species with presence of USUV/WNV (long shot)
2. Isolate insect-specific virus for characterization and vector competence studies in laboratory-held Culex pipiens
Outline anti-viral immune response of Culex pipiens
1. Acquire list of Toll, IMD and/or JAK-STAT downstream target genes in Culex quinquefasciatus HSU and Culex Tarsalis CT cells
2. In vivo RNAi knock-down of Toll, IMD & JAK-STAT in laboratory-held Culex pipiens

Paper 1: The Culex transcriptome of the Toll, IMD & JAK-STAT immune signalling pathways

Paper 2: A role for the Toll/IMD/JAK-STAT pathway in the immune response of Culex pipiens to WNV and USUV

Paper 3: Novel Culex pipiens insect-specific virus restricts WNV infection

Timeline

Tags matching with the contents of track 14/24

Methods

Labwork
- PCR: Presence of USUV & WNV in field-caught Dutch Culex pipiens will be determined with PCR. PCR also used for regular laboratory work.
- RNA/DNA extraction: RNA (and DNA) will be isolated from mosquito samples for RNA-seq (virome analysis) and PCR. Similar work for mosquito cell-lines.
- In vivo: The immune response of Culex pipiens to arboviruses will be assayed in laboratory-held mosquito colony in Wageningen. Vector competence studies may also be performed with isolated insect-specific viruses.
- In vitro: Culex cell-lines will be used for determining the transcriptome of the Toll, IMD and/or JAK-STAT pathway. Cell-lines also for culturing of viruses from field-caught mosquitoes.
- Next generation sequencing (NGS): RNA-seq will be used for detection of insect-specific viruses in Dutch mosquito samples and to determine the transcriptome in response to Toll, IMD or JAK-STAT pathway activation.
- Molecular Cloning: Cloning work will be performed to create Ago2-KO cell-lines and in vitro reporter assays for immune pathway activation.
- Plaque Assay: For in vivo studies and in vitro virus culture, plaque assays will be used (or equivalent techniques) to determine virus presence and amount.
Fieldwork
- Mosquito trapping: Mosquitoes caught by trapping will be analysed for arboviral presence and virome analysis.
Literature
- Literature review: Review will be written to provide an overview of anti-viral immunity genes in mosquito species and Drosophila.

Topics

Vector:
- Vector immune response: Vector immune response will be tested on laboratory-held Culex pipiens and interventions in the immune signalling pathways will be performed to investigate their role in the immune response.
- Vector susceptibility: Vector susceptibility/Vector competence studies will be performed in the acquirement of an insect-specific virus from Dutch Culex pipiens to see potential for intervention.
- Vector competence: Vector susceptibility/Vector competence studies will be performed in the acquirement of an insect-specific virus from Dutch Culex pipiens to see potential for intervention.
Virus:
- Virus-mosquito interaction: The impact of viruses (both arboviruses and insect-specific viruses) on the mosquito immune response will be analysed in laboratory-held Culex pipiens colony.
Interventions:
- Other interventions: In the mapping of the virome for Dutch Culex pipiens, we hope to acquire an insect-specific virus that can be used to restrict vector competence of arboviruses.

Species

Species:
- Mosquito: The project looks at arboviral presence and naturally-infecting viruses in Dutch Culex pipiens and the Culex pipiens anti-viral immune response.

Virus

Virus:
- Virus (general): This project will analyse the Dutch Culex pipiens samples for the presence of all viruses deemed relevant within the One Health pact.
- West Nile virus: Presence of WNV in Dutch Culex pipiens will be determined in this project. WNV will also be used as a model arbovirus when looking at the anti-viral immune response and the effect of insect-specific viruses on vector competence.
- Usutu Virus: Presence of USUV in Dutch Culex pipiens will be determined in this project. USUV will also be used as a model arbovirus when looking at the anti-viral immune response and the effect of insect-specific viruses on vector competence.

Tom van der Most