Methods
Lab work
Polymerase Chain Reaction.
1 Rody Blom: qPCR is used to biotype Culex pipiens mosquitoes. In addition, I will use qPCR assays to identify vertebrate host origin from mosquito blood meals.
2. Nnomzie Atama: PCR will be used for viral detection in live and dead birds sampled.
3. Tjomme van Mastrigt: Co-infection with avian malaria.
4. Jurrian van Irsel: My research involves PCR to detect Usutu and WN virus. Besides, I will use some novel methods that are nowhere described related to immune assays.
5. Chiara de Bellegarde: PCR will be used to detect arbovirus infected travellers upon their return.
13. Charlotte Linthout: I use qPCR to identify the biotype of the Cx pipiens complex (pipiens and molestus) to set up a new colony to use it later for my experiments.
14/24. Jurgen Moonen: Canbe used to study the expression of immunity related genes (quantitative PCR).
14/24. Tom van der Most: Presence of USUV & WNV in field-caught Dutch Culex pipiens will be determined with PCR. PCR also used for regular laboratory work.
15. Sam Boerlijst: Mosquito species identification as species complexes can be hard to separate morphologically.
16. Gianfilippo Agliani: I will use PCR to detect viral RNA in tissue samples.
17. Imke Visser: Virus detection in tissues and cell culture
18. Eleanor Marshall: Virus detection in tissues and cell culture
19. Tessa Nelemans: We will use this technique to build the infectious clone and develop expression constructs of the viral proteins etc.
20. Muriel Aguilar Bretones: Amplification of complementary determining regions of antibodies.
25. Jo Duyvestyn: PCR willbe used in amplification of samples, confirmation of cloning, and (incomplete sentence).
26 Joyce van Bree: Amplification viral genomic fragments, cloning.
Enzyme-linked immunosorbent assay, used for detecting and quantifying soluble substances such as peptides, proteins, antibodies.
2. Nnomzie Atama: Serological monitoring of birds and wild mammals for circulation of VBD.
3. Tjomme van Mastrigt: Immunological assays will be using this technique.
8. Kiki Streng: I will use ELISA tests as a screening method for determining the baseline seroprevalence of several arboviruses in multiple animal species.
17. Imke Visser: Antibody detection upon in vivo virus infection with/without mosquito saliva.
20. Muriel Aguilar Bretones: Custom and multiplex readout of quantifying antibody reactivity will be setup.
14/24. Jurgen Moonen: Can be used to look at factors involved in immunity on the protein level.
Discover and functionally characterize the individual antibody clones that are generated upon vaccination or infection.
20. Muriel Aguilar Bretones: This is the main technical aim of my project and may complement studies on other components of the immune response, immune pathogenesis, virulence, vaccine efficacy and safety
The purification of RNA or DNA from biological samples.
1. Rody Blom: DNA extraction from mosquitoes and/or blood meals for subsequent biotyping and blood meal analyses.
2. Nnomzie Atama: For extracting RNA/DNA for my PCRs.
3. Tjomme van Mastrigt: Avian host genetic diversity may be linked to prevalence in the wild, and where possible through collaboration in infection trials.
14/24. Jurgen Moonen: Essential for many different wet lab approaches.
14/24. Tom van der Most: RNA (and DNA) will be isolated from mosquito samples for RNA-seq (virome analysis) and PCR. Similar work for mosquito cell-lines.
13. Charlotte Linthout: I have to extract DNA from mosquitoes to do the qPCR (see above) I will have to work with RNA extraction in the future as soon as I start with infected mosquitoes to see the infection, dissemination and transmission rates.
16. Gianfilippo Agliani: RNA extraction from tissue for detection of virus by PCR.
17. Imke Visser: RNA extraction from tissue samples to determine viral load.
18. Eleanor Marshall: I may be extracting RNA to determine viral presence in tissues.
19. Tessa Nelemans: This technique will be used to obtain RNA from infected cells or the ex vivo samples for qPCR or transcriptomics.
20. Muriel Aguilar Bretones: Used to study B-cell receptor sequences and virus genome copies.
25. Jo Duyvestyn: Purifying infectious clone or expression construct plasmids from growth cultures, or RNA from cell supernatants when growing virus or clones.
26 Joyce van Bree: Extraction of viral RNA to generate infectious clones, but also from samples to determine viral gRNA copies
The reduction in viral infectivity by the binding of antibodies to the surface of viral particles (virions), thereby blocking a step in the viral replication cycle that precedes virally encoded transcription or synthesis.
2. Nnomzie Atama: Used for serological detection in birds and wild mammals.
3. Tjomme van Mastrigt: I will be using viral neutralization assays to identify patterns in migratory birds throughout the annual cycle.
8. Kiki Streng: These tests will be used as confirmatory tests if screening ELISA tests have a positive result.
17. Imke Visser: Perhaps after detecting antibody upon in vivo virus infection with/without mosquito saliva (see above), determine whether these antibodies are neutralizing.
20. Muriel Aguilar Bretones: Part of functional analysis of antibodies.
25. Jo Duyvestyn: Will measure neutralizing antibody titres of vaccines that we test in animal models.
Research performed in living animals/humans.
3. Tjomme van Mastrigt: Living birds in the wild will be studied by repeated sampling for arboviruses throughout the annual cycle. Mosquitoes will be studied in vivo to learn about mosquito feeding preferences in an experimental setting.
14/24. Jurgen Moonen: I will work with mosquitoes.
14/24. Tom van der Most: 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.
13. Charlotte Linthout: Working with Mosquitoes.
16. Gianfilippo Agliani: I will perform experimental infection of live birds and mice to study viral pathogenesis.
17. Imke Visser: To identify the dose and route through which the vertebrate host is most efficiently infected; to elucidate the mechanisms by which mosquito saliva from different species differentially contributes to the transmission, enhancement, and dissemination of distinct viruses to and within the vertebrate host; to determine the minimal level of host viremia contributing to maintaining a transmission cycle; to investigate non-vector borne routes of viral infection.
18. Eleanor Marshall: I will do a lot of in vivo work to determine routes and spatio-temporal kinetics of neuroinvasion in rodents.
20. Muriel Aguilar Bretones: Antibodies characterized in our studies may be used in animal models to address their clinical impact. This research will be performed in collaboration.
25. Jo Duyvestyn: IFNAR-/- mouse models to look at pathogenesis of mutant USUV clones, and vaccine testing.
Experimental studies with live mosquitoes in their (simulated) natural habitat, such as living lab experiments. All life stages of the mosquitoes fall under this.
3. Tjomme van Mastrigt: In vitro work will be done during immunological assays to challenge the immune system.
5. Marianne Rijtma: I will use blood samples to measure the innate immune response.
14/24. Jurgen Moonen: I will work with mosquito cells.
14/24. Tom van der Most: 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.
17. Imke Visser: Develop in vitro models to investigate the mechanisms by which mosquito saliva from different species differentially contributes to the transmission, enhancement, and dissemination of distinct viruses to and within the vertebrate host using relevant (human and bird) cell lines.
18. Eleanor Marshall: I will develop in vitro models of neuroinvasion using primary cell cultures.
19. Tessa Nelemans: We will be using different cell lines to assess viral titers, growth kinetics and the innate immune response.
20. Muriel Aguilar Bretones: Primary B-cells will be analysed in depth using in vitro culture systems.
25. Jo Duyvestyn: Range of cell lines models including Vero/BHK to grow/optimize virus, immune competent human cell lines to study immune evasion and pathogenesis of mutants, mosquito cell lines including C636 (and a culex line?) for characterisation of mutants.
26 Joyce van Bree: Titrations, expression of tagged-proteins.
The use of modern sequencing techniques to determine the sequence of nucleotides in a section of RNA/DNA.
1. Rody Blom: Sequencing of blood meal qPCR products to identify blood meal origin.
2. Nnomzie Atama: For comparison of phylogenetic relationship across strains of viral particles detected in wildlife to determine possibilities for introduction.
14/24. Jurgen Moonen: 1) To study the virome of the mosquitoes. 2) To perform RNAseq to study mosquito immunity on a large scale
14/24. Tom van der Most: 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.
13. Charlotte Linthout: Will use in the future, not sure yet for which experiment but will clarify this later.
15. Sam Boerlijst: Mosquito and predator species identification as species complexes can be hard to separate morphologically; culicid eDNA shedding patterns.
19. Tessa Nelemans: We have used NGS to sequence our USUV stocks.
20. Muriel Aguilar Bretones: NGS of B-cell receptor analysis could be combined with in vitro antigen-specific analysis of clones.
25. Jo Duyvestyn: To confirm the sequences of our clones and mutants.
Using vectors containing replicates of genes or the entire virus in order to amplify and manipulate the DNA.
14/24. Jurgen Moonen: I will probably use this technique during my PhD.
14/24. Tom van der Most: Cloning work will be performed to create Ago2-KO cell-lines and in vitro reporter assays for immune pathway activation.
19. Tessa Nelemans: We will build the infectious clone and develop expression constructs of the viral proteins etc.
20. Muriel Aguilar Bretones: Variable regions of B-cell receptors will be cloned and expressed to generate recombinant antibody clones.
25. Jo Duyvestyn: To create an USUV infectious clone and expression.
26 Joyce van Bree: Generate CpG/UpA viral mutants, tagging proteins.
Studies done on cells or tissue extracted from an organism, and used in an external environment with minimal alteration of natural conditions.
3. Tjomme van Mastrigt: Work will be done during immunological assays to challenge the immune system.
13. Charlotte Linthout: Will probably work with mosquito cell lines.
16. Gianfilippo Agliani: I will perform ex vivo experiments to study virus-cell interactions.
17. Imke Visser: Ex vivo skin tissue for viral infection experiments with/without mosquito saliva.
18. Eleanor Marshall: I may use ex vivo brain tissue for viral infection experiments.
19. Tessa Nelemans: We will use an ex vivo skin model to assess the innate immune response after an arbovirus infection.
20. Muriel Aguilar Bretones: Immune cells from peripheral blood or target tissues will be phenotypically characterized.
25. Jo Duyvestyn: As a model to study pathogenesis of mutant constructs, likely in collaboration.
A method for measuring the titre of infectious virus using serial dilutions onto susceptible cell lines.
2. Nnomzie Atama: For determining the infectivity and role of wild species as reservoirs or amplifying hosts.
3. Tjomme van Mastrigt: Work will be done during immunological assays to challenge the immune system.
14/24. Jurgen Moonen: Will probably be part of the virus work that I will be doing.
14/24. Tom van der Most: For in vivo studies and in vitro virus culture, plaque assays will be used (or equivalent techniques) to determine virus presence and amount.
16. Gianfilippo Agliani: Virus titration from tissues (can also be by TCID50).
17. Imke Visser: Either plaque assays or TCID50 will be used to determine viral titers of in vitro and in vivo infection experiments.
18. Eleanor Marshall: I will use plaque or TCID50 assays to determine titres of virus in infection experiments.
19. Tessa Nelemans: We use plaque assays to determine the viral titre of our virus stocks.
20. Muriel Aguilar Bretones: Part of functional analysis of antibodies.
25. Jo Duyvestyn: Will be used to characterize WT, infectious clone, and mutant USUV (and other?) viruses.
26 Joyce van Bree: Determine plaques from mutant viruses vs wt viruses.
Experimental studies with live mosquitoes in their (simulated) natural habitat, such as living lab experiments. All life stages of the mosquitoes fall under this.
1. Rody Blom: I will do trapping experiments in a flight cage. I will do this to 1) test the BG Counter and 2) assess the efficacy of molasses-fermentation based trapping.
3. Tjomme van Mastrigt: Mosquitoes will be studied in vivo to learn about mosquito feeding preferences in an experimental setting.
13. Charlotte Linthout: I will infect different mosquito species (Ae albopictus, Ae japonicus and Cx pipiens biotypes) with viruses to investigate their vector competence under different (temperature) scenario’s. I will also work with mosquito larvae and expose them to different stress factors in order to test their vector competence when they are adults.
15. Sam Boerlijst: Microcosm/Mesocosm experiments.
17. Imke Visser: I may use mosquito saliva from mosquitoes reared under living lab conditions (collab. with Maarten Schrama, LU) if possible. I will use live (uninfected or experimentally infected) mosquitoes to be used for transmission studies (collab. with Sander Koenraadt/Gorben Pijlman, WUR).
26 Joyce van Bree: Effect of introducing CpG/UpA in viral genomes on transmission by mosquitoes.
Experimental studies with live animals & birds, for example to study behavior and clinical symptoms.
2. Nnomzie Atama: Pathogenicity of viruses in lab models will be conducted.
3. Tjomme van Mastrigt: Behavioural studies will be done using GPS transmitters on wild birds to study their habitat use in urban and rural settings, and to characterize host movements that may be important in the spread of vector-borne diseases.
16. Gianfilippo Agliani: I will perform experimental infection of live birds and mice to study viral pathogenesis.
17. Imke Visser: To identify the dose and route through which the vertebrate host is most efficiently infected; to elucidate the mechanisms by which mosquito saliva from different species differentially contributes to the transmission, enhancement, and dissemination of distinct viruses to and within the vertebrate host; to determine the minimal level of host viremia contributing to maintaining a transmission cycle; to investigate non-vector borne routes of viral infection.
18. Eleanor Marshall: As above within vivo: I will also do behavioural studies to see whether function of the brain is compromised, at what stage of infection and where the virus can be found in the brain.
20. Muriel Aguilar Bretones: Antibodies characterized in our studies may be used in animal models to address their clinical impact. This research will be performed in collaboration.
Fieldwork
Catching individual mosquitoes by using a mosquito trap.
1. Rody Blom: I will do small and large scale mosquito field surveys.
3. Tjomme van Mastrigt: Mosquitoes will be captured only for use in experimental settings. Different habitats will be chosen to compare feeding preferences of mosquitoes in a standardized environment.
7. Ayat Abourashed: I will have students create their own mosquito traps and collect mosquitoes for other PhD tracks.
8. Kiki Streng: Mosquitoes will be trapped at livestock barns to determine presence and abundance.
13. Charlotte Linthout: I collected Culex egg rafts to set up a new colony and in Lelystad I am collecting Aedes japonicus mosquitoes during summer (June-September) to use in my experiments.
14/24. Tom van der Most: Mosquitoes caught by trapping will be analysed for arboviral presence and virome analysis.
15. Sam Boerlijst: Collection for use in experiments/ investigation of oviposition choice.
Characterizing all physical attributes that influence or sustain organisms within a habitat.
3. Tjomme van Mastrigt: Bird nests will be collected and ectoparasite loads will be quantified to identify effects on the rearing environment on the immunocompetence of nestling birds.
4. Jurrian van Irsel: I will use some habitat characterization based on existing land-use data to determine the influence on Usutu occurrence.
Observing bird presence and/or catching live or dead birds and/or taking samples from live or dead birds.
2. Nnomzie Atama: As part of our surveillance plan for VBD in live and dead birds.
3. Tjomme van Mastrigt: Wild birds will be caught on their breeding grounds as well as on stop-over locations during migrations to characterize annual cycle variation in immunocompetence and to link this to parasite pressure. Moreover, resident birds will be caught to study immune function in a natural context.
16. Gianfilippo Agliani: I will be involved in dead bird sampling for detection of USUV/WNV.
21. Pauline de Best: Within my project, knowledge will be translated into public health action, Bird samples and surveillance outcomes are a part of this.
4. Jurrian van Irsel: My research involves trapping birds for surveillance and experimental work on Blackbird nestlings in nests in the field.
Observing wildlife presence and/or catching live or dead wildlife and/or taking samples from live or dead wildlife.
2. Nnomzie Atama: Surveillance and monitoring plans.
8. Kiki Streng: Samples of large ruminants, equines and potentially zoo animals will be used in a seroprevalence study.
16. Gianfilippo Agliani: I will be involved in dead wildlife sampling for detection of USUV/WNV.
21. Pauline de Best: Within my project, knowledge will be translated into public health action, wildlife samples and surveillance outcomes are a part of this.
Observing domestic animal presence and/or catching live or dead domestic animals and/or taking samples from live or dead domestic animals.
8. Kiki Streng: Samples of livestock, equines and pets will be used in a seroprevalence study.
21. Pauline de Best: Within my project, knowledge will be translated into public health action, domestic animal samples and surveillance outcomes are a part of this.
The ongoing, systematic collection, analysis, and interpretation of health-related data essential to planning, implementation, and evaluation of public health practice.
6. Chiara de Bellegarde: Human serum samples will be tested to explore seropositivity for vector-borne diseases in the Netherlands and after travelling in Europe.
7. Ayat Abourashed: I will combine the data collected using Mosquito Alert with traveler data (Track 6) to analyze trends.
20. Muriel Aguilar Bretones: Peripheral blood mononuclear cells of arbovirus vaccinees or infected individuals are required for our research.
21. Pauline de Best: Within my project, knowledge will be translated into public health action, public health surveillance outcomes are a part of this.
Citizen science
Scientific research conducted by non-professionals, public participation.
1. Rody Blom: I use citizen science (Muggenradar) to collect nuisance data and mosquito specimens. This data will be compared to the data collected in the field.
3. Tjomme van Mastrigt: Experienced and well-trained volunteer bird ringers will contribute to the arbovirus surveillance in wild bird populations throughout the Netherlands as well as abroad.
4. Jurrian van Irsel: All bird presence data (Sovon data) and the live surveillance bird ringing data (VT data) are collected by CS.
6: Chiara de Bellegarde: Travelers will be asked to keep a digital symptom diary during their journey and provide information on potential risk behaviors. In addition, they might be asked to collect data on the mosquito population in their travel destinations.
7. Ayat Abourashed: I will create a program where students will collect data regarding mosquitoes and breeding sites using Mosquito Alert. Students may collect other data for other PhD projects.
21. Pauline de Best: Outcomes collected by citizen scientists could be translated into public health actions. Interventions will involve citizen actions.
Modelling
The use of computer algorithms to predict the distribution of a species across geographic space and time using environmental data (also known as: environmental/ecological niche modelling, habitat modelling, predictive habitat distribution modelling, and range mapping).
3. Tjomme van Mastrigt: spatial aspects in population dynamics may be taken into account when this turns out to be essential in population modelling of avian host dynamics.
4. Jurrian van Irsel: I will make distribution models of Usutu occurrence and try to develop a species distribution model based on current annual bird monitoring surveys.
5. Marianne Rijtma: I will use environmental niche models for birds and mosquitoes to map their distribution to use in predicting spatiotemporal variation in WNV risk.
7. Ayat Abourashed: I will use the data collected via Mosquito Alert to create predictive models for certain mosquito species distributions.
8. Kiki Streng: To estimate the risk of introduction or spread among (domestic) animals, species distribution maps will be developed.
11. Clara Delecroix: We will use various models for analysis.
21. Pauline de Best: Identified risks/ tipping points and other outcomes can be translated into public health actions.
(27). Martha Delar: Working with PhD12 to build mosquito model. May also look at modelling the distribution of various host species (if not done as part of other projects).
The construction and analysis of mathematical models based on epidemiological data that predict the spread of a disease through a population.
2. Nnomzie Atama: Data generated from bird and wild animal surveillance will be used in models to predict disease spread.
3. Tjomme van Mastrigt: Transitions between infection states will be estimated from capture-mark recapture models, and will be incorporated in integrated approaches in collaboration with other PhD students.
4. Jurrian van Irsel: Partly including this, as I will be determining the effect of bird community and species host susceptibility on the R0.
6. Chiara de Bellegarde: Epidemiological models will be used to assess the contribution of travelling to the importation of vector-borne diseases.
7. Ayat Abourashed: I will combine the data collected using Mosquito Alert with traveller data (Track 6) to create predictive epidemiological models.
9. Mariken de Wit: I will build transmission models using collected data to predict spread (R0), speed and final size of potential outbreaks.
10. Afonso Dimas Martins: I will be developing and analyzing eco-epidemiological ODE models that incorporate the viral cycles as well as information from the ecosystem (competition, predation, etc.).
11. Clara Delecroix: We will use various models for analysis.
17. Imke Visser: I will determine the minimal level of host viremia contributing to maintaining a transmission cycle, which will give insight into the spread of a virus through a population considering the amount of virus present in a patient needed for a biting mosquito to become infected and subsequently be able to infect other people 21.
21. Pauline de Best: Identified risks/ tipping points and other outcomes can be translated into public health actions.
The construction and analysis of mathematical models of ecological processes.
3. Tjomme van Mastrigt: Survival and antibody dynamics will be studied in the context of capture-mark recapture studies of wild birds on ringing sites to build statistical models that allow multiple sources of non-randomly missing data and uncertainty to be taken into account in inference of demographic and epidemiological parameters.
5. Marianne Rijtma: I will use a statistical model to identify bird species associated with WNV outbreaks.
10. Afonso Dimas Martins: I will be developing and analyzing eco-epidemiological ODE models that incorporate the viral cycles as well as information from the ecosystem (competition, predation, etc.).
11. Clara Delecroix: We will use various models for analysis.
21. Pauline de Best: Identified risks/ tipping points and other outcomes can be translated into public health actions.
(27) Martha Delar: Working with PhD12 to build mosquito model. Will also potentially be creating (or using existing) models for ecosystem processes to predict how things will change in the future.
Literature
An overview and critical analysis of what has been published on a topic by accredited researchers.
1. Rody Blom: I am currently working on a literature review on urbanization and mosquito-borne disease outbreak risk. Primarily from an entomological and ecological point of view.
2. Nnomzie Atama: Working on a literature review of the wild animal hosts for VBD other than wild birds.
3. Tjomme van Mastrigt: A meta-analysis on the published prevalences will be used to quantify USUV and WNV prevalence across avian taxa, special attention will be paid to sources of bias arising from contrasting methodologies in published data.
6. Chiara de Bellegarde: I will be performing a literature review on arbovirus infections in humans in Europe.
7. Ayat Abourashed: I will complete a literature review to determine what citizen science programs (focusing on life sciences) have been implemented at schools.
13. Charlotte Linthout: I will do this towards the end of my PhD.
14/24. Jurgen Moonen: I hope to write a literature review about antiviral immune pathways of Aedes albopictus.
14/24. Tom van der Most: Review will be written to provide an overview of anti-viral immunity genes in mosquito species and Drosophila.
15. Sam Boerlijst: Defining knowledge lacune.
16. Gianfilippo Agliani: I performed a review of the literature concerning lesions on the CNS of animals, induced by mosquito-borne flaviviruses.
17. Imke Visser: I am writing a literature review on the transmission of arthropod-borne viruses from vector to host and the effect of vector saliva on viral transmission, pathogenesis, and the host immune system.
18. Eleanor Marshall: I am currently writing a literature review on what is known about the neuroinvasive strategies used by arboviruses.
20. Muriel Aguilar Bretones: Analysis of literature is an integral part of my project. However, so far a review manuscript is not planned.
25. Jo Duyvestyn: Likely will write this on comparing virulence factors of flaviviruses in a vaccine design context.
21. Pauline de Best: I will likely conduct a literature review on the different types of vector control (integrated vector management) and their applicability to the Dutch situation.
26. Joyce van Bree: Not sure whether I will write one soon.
Document analysis is a form of qualitative research in which documents are interpreted by the researcher to give voice and meaning around an assessment topic. Analyzing documents incorporates coding content into themes similar to how focus group or interview transcripts are analyzed.
6. Chiara de Bellegarde: I will be performing document analysis of technical documents such as epidemiological reports and rapid risk assessments.
21. Pauline de Best: I will perform a document analysis to identify stakeholders and learn lessons from guidelines of other countries dealing with arboviruses.
26. Joyce van Bree: Not sure.
Qualitative research
Qualitative research techniques which involve intensive conversations with a small number of respondents to explore their perspectives on a particular idea, situation or process.
8. Kiki Streng: I will perform interviews to investigate expert opinion on possible/most likely future scenarios of the agricultural sector (with emphasis on livestock farming).
21. Pauline de Best: I will perform interviews/ focus groups with stakeholders to identify additional stakeholders, the role of stakeholder within the preparedness and response and to identify prioritized research outcomes for outbreak preparedness and control.
A process or action research methodology used to explore the various opinions that different stakeholders may have on potential outcomes, control measures or infectious disease response and their relative influence.
21. Pauline de Best: I will perform stakeholder analysis to identify all the relevant stakeholders in the field of arboviral preparedness and response.