Tessa
Nelemans

Project

Track: 19, Role of host innate immune responses and arbovirus innate

Tags

Promotor

Prof. Eric Snijder

Co-promoter(s)

Dr. Martijn van Hemert and Dr. Ir. Marjolein Kikkert

Institute(s)

Leiden University Medical Centre

E-mail

Telefoon

+31 (0) 624171986

Description PhD project

The Netherlands is particularly vulnerable to outbreaks of infectious diseases due to climate change, the water-rich environment, the dense population and globalization. The main focus of this track will be on emerging vector-borne viruses including West Nile virus (WNV) and Usutu virus (USUV). WNV (lineage 2) currently affects South-Eastern Europe, though several cases have now been reported in Germany as well. USUV already circulates in the Netherlands and is largely associated with mortality in birds, nevertheless some human cases of USUV-associated neuroinvasive disease have been reported. The innate immune system functions as the first line of defense against invading pathogens including these arboviruses. In order to facilitate their replication in the host cell, many viruses have developed mechanisms to counteract the innate immune response. This project aims to identify the key host innate immune pathways and the arbovirus evasion mechanism involved in transmissibility and disease outcome. This knowledge might aid in predicting virulence factors of new emerging arboviruses and may be used in the development of live-attenuated vaccines.

For USUV it is largely unknown what kind of evasion mechanisms are active during infection. This project will identify the viral proteins most capable of antagonizing the interferon response. Moreover, we will assess the interaction of these viral proteins with host factors and try elucidate the molecular mechanisms behind their immune evasion activity. We will also develop a reverse genetics system in order to be able to introduce attenuating mutations in the virus genome and study its effect onthe host innate immune response. Lastly, we want to study the dynamics of an arbovirus infection in an ex vivo skin model.


Research questions / objectives

  1. Determine the key viral proteins involved in evasion of the host innate immune response and determine attenuating mutations.
  2. Developing full-length infectious cDNA clones of USUV and construction of arboviruses deficient in their immune evasion activity.
  3. Determine the molecular mechanism of immune evasion activities of USUV and WNV.
  4. Examine the host innate immune response to USUV and WNV in the ex vivo skin model.

Timeline


Tags matching with the contents of track 19

Methods

  • Labwork

    • PCR: We will use this technique to build the infectious clone and develop expression constructs of the viral proteins etc.
    • RNA/DNA extraction: This technique will be used to obtain RNA from infected cells or the ex vivo samples for qPCR or transcriptomics.
    • In vitro: We will be using different cell lines to assess viral titers, growth kinetics and the innate immune response.
    • Next generation sequencing (NGS): We have used NGS to sequence our USUV stocks.
    • Molecular Cloning: We will build the infectious clone and develop expression constructs of the viral proteins etc.
    • Ex Vivo: We will use an ex vivo skin model to assess the innate immune response after an arbovirus infection.
    • Plaque Assay: We use plaque assays to determine the viral titer of our virus stocks.

Topics

  • Virus:

    • Pathogenicity: We will introduce mutations in the viral genome that will block its immune evasion activity and will attenuate the virus. The pathogenicity of attenuated viruses will be tested in the ex vivo model and in vivo (in collaboration with track 25)
    • Virus-host interaction: We will assess the interaction of viral proteins with host factors to uncover the molecular mechanism of the viral immune evasion activity.

Species

  • Species:

    • Human: Ex vivo skin model will be human derived

Virus

  • Virus:

    • West Nile virus: The main focus of this project will be on WNV and USUV.
    • Usutu Virus: The main focus of this project will be on WNV and USUV.

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One Health PACT with project number 109986 is (partly) financed by the Dutch Research Council (NWO).