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Current Projects

Infection models and studies

a)

Vector competence of Mosquitoes and ticks: We establish an infection system to analyse the vector competence of German mosquitoes for arboviruses in our laboratory by using field-collected immature stages reared until the adult stage (Figure 2). Susceptibility to viral pathogens is measured using standard vector competence indices: infection, dissemination and transmission. Thus far, we have analysed the susceptibility of German Culex (3) and Aedes (4) species for WNV as well as performing comparative analyses with Culex and Aedes species from Switzerland (5). Within the following years we aim to expand the infection experiments to other mosquito species and arboviruses (DFG-ECOVIR) as well as insect-specific viruses (BMBF-iMINION). Ultimately, we will also use this knowledge to develop biological intervention strategies (DFG-EVICTION). Similar to mosquito infection systems, we have also established feeing protocols for ticks with tick-borne viruses (7). In the projects VECTORS (DFG) and TBENAGER, we aim to characterise the factors driving TBEV transmission and emergence. These studies include virome and microbiome analysis of tick populations and their influence of TBEV transmission by those ticks as well as specific adaptations of virus isolates to tick populations.

 

b)

Drosophila models: Using the well-characterised insect model Drosophila melanogaster, we were able to unravel several underlying immune mechanisms (7-11 and Figure 3). Within our project ANTIGONE (DFG) we will use this model and mosquito infection systems (7) to analyse the role of small RNA interference pathways on transovarial transmission and the influence of immune pathways on neuronal activity (Figure 4) and behavioural changes following an arbovirus infection.

 

 

 

 

Figure 3: Intrinsic factors that interfere with the vector competence of mosquitoes.

The vector competence of a certain mosquito species is characterized by several factors. Firstly, the ability of the virus to overcome the midgut. Secondly the ability of the virus to replicate in various tissues of the insect host and most importantly the efficient dissemination of infectious viral particles to the saliva. The virus replication in the mosquito midgut is regulated by the gut microbiota represented by the Wolbachia endosymbiont (1) which can interfere with the virus replication by various ways. The virus replication in different tissues can trigger several antiviral pathways such as the RNAi pathways represented by Dicer cleavage and the inducible immune responses represented by the induction of Vago (2).

 

Figure 3 with figure legend were taken from: Schulz& Becker, Mosquitoes as arbovirus vectors: from species identification to vector competence. In: Mosquito-borne diseases: implications for public health. Parasitology Research Monographs, Band 10; Cham, Switzerland: Springer, 2018, 163-212.

 

 

 

 

Figure 4: Electroantennogram setup for Drosophila and mosquitoes to measure neuronal activity after different olfactory stimuli in infected and non-infected state.

 

 

c)

Commensal bacteria and viruses: One huge influence on behaviour and the competence to transmit viral pathogens is the microbiome and virome of vectors. The Culex pipiens mosquitos forms an interesting tripartite relationship with the endosymbiotic bacterium Wolbachia pipientis and the bacteriophage WO infecting Wolbachia. Others and we have addressed the question of Wolbachia distribution and influence on infection resistance in mosquitoes (12). First studies in German Culex pipiens molestus have shown that different population harbour Wolbachia. Although all mosquito strains were positive for Wolbachia pipientis, the outcome of Wolbachia infection on co-infection with West Nile Virus and blood-feeding behaviour were different (Figure 5). The in-depth characterisation of WO-Phage genotypes revealed differences regarding the WO phage colonising the bacteria in different Culex pipiens strains. The underlying mechanisms are still unclear but will be targeted in future projects (Collaboration with Institute for Pathology).

 

 

 

 

Figure 5: Wolbachia-Virus-mosquito interactions
Schematic representation of a mosquito with Wolbachia infection are shown as orange dots and infecting WNV shown as yellow dots. The midgut is shown in red, the salivary gland system in blue, and the central nervous system in green.
(A) Wolbachia infection of the midgut blocking WNV infection. (B) Wolbachia infection of the central nervous system influencing feeding behaviour. (C) Wolbachia negative control

TBENAGER

Vectors distribution and specification:

 

Tick-Borne ENcephAlitis in GERmany (TBENAGER): Influence of population based differences in ticks and bank voles on TBEV infection and transmission cycles in the natural foci
Duration:    2017-2022
Funding bodies    BMBF Nationales Forschungsnetz zoonotische Infektionskrankheiten

 

As the spread of arboviruses is dependent on the presence of a suitable mosquito vector, the knowledge of the mosquito species distribution, genetic diversity of mosquito populations’, colonisation pattern and vector competence of these mosquitoes is crucial for the risk estimation. The first critical issue for mosquito surveillance programs in Germany is the exact classification of species and population dynamics. To facilitate detection of different Culex species and to analyse genetic diversity and colonialization pattern of invasive species, we established a multiplex qPCR (1) and microsatellite techniques (2). By using these techniques, we will continue our research on mosquito species distribution and population genetics and expanded our projects to Madagascar (Collaboration with Prof.. Strube, manuscript in preparation), Palestine (Collaboration with Prof.. Strube, manuscript in preparation) and West African countries (DFG-ECOVIR). Furthermore, we participate in network projects to analyse the distribution and population genetics of Ixodes ticks in Germany (BMBF-TBENAGER).

 

Figure 2: Graphical representation of vector competence assay 

The analysis of resident mosquito populations for virus presence and vector competence for the respective virus starts with the collection of mosquitoes (1). Subsequently, the mosquitoes are subjected to morphological taxonomic classification (2) and are pooled according to species and location. Mosquito pools are homogenized to isolate nucleic acids for PCR and proteins for MALD-TOF MS. These data are used for taxonomic confirmation (3) and abundance statistics (4) or virus screening. Virus positive pools will be used for virus isolation (5) which can then be used for vector competence assays via oral infection (9). To obtain mosquito samples for vector competence assays, eggs of resident mosquito populations are collected (6) and reared in the laboratory (7). From each larval culture, some specimens will be used for taxonomic identification (8). Larvae from the same location and species are pooled and emerging adult females will be used for vector competence assays. New virus isolates are mixed with blood and fed to 4-7 days old female mosquitoes (9). After different times of infection some mosquitoes are sacrificed and body infection rates (IR), dissemination rates (DR) and transmission rates (TR) will be measured by virus titration (10).

 

Figure 2 with figure legend were taken from: Schulz& Becker, Mosquitoes as arbovirus vectors: from species identification to vector competence. In: Mosquito-borne diseases: implications for public health. Parasitology Research Monographs, Band 10; Cham, Switzerland: Springer, 2018, 163-212.

 

 See also "Infection models and studies" for further information.

VIPER; N-RENNT; VECTORS

Virus detection, pathogenesis and intervention (VIPER): Influence of genome reassortment of mosquito-borne Peribunyaviridae on the interaction with host innate immune response
Duration:    2019-2023
Funding bodies    DFG Graduate School (GRK 2485)

 

Vector competence for TBEV replication limiting viral spread (VECTORS)
Duration:    2017-2020
Funding bodies    DFG  in the frame of  the SPP "Ecology and species barriers in emerging viral diseases "

 

Viromes and viral evolution:

 

In nature, viruses are the most abundant reproductive units, and are present in nearly all habitats (aquatic, sediment soils), and organisms (archaea, bacteria, protozoa and higher animals) sampled thus far. Related to arbovirus transmission, it is of particular interest if vector-associated viruses can interfere with arbovirus transmission or by themselves harbour the potential to cause infections in humans and livestock. To address this question we have recently started to sequence the virome of tick in Lower Saxony. First results hint to a large variety of tick-associated new viruses whose potential to infect humans or to interfere with TBEV transmission is not explored yet (DFG-VECTORS-2). For further information also see "Infection models and studies".

 

 

 

Figure 6: Virus reassortment
The parental viruses 1 (blue) and 2 (red) infect the same host cells. After replication of the viral genome segments new viruses can take mixes of parental segments creating new reassortant viruses shown below (grey)

 

Furthermore, Viruses are regularly exposed to hostile conditions either in the environment or by the immune system of their hosts, which seeks to eliminate viral infection. In consequence, viral pathogens have evolved mechanisms to evade the immune system and infect new hosts/vectors and to ensure environmental stability. The rapid adaptation of viruses to new environments and conditions is favoured by the high mutation rate of viral genomes and recombination and reassortation (Figure 6). The latter is most common for segmented RNA viruses and grants a high degree of genome plasticity to those viruses. Genetic reassortment has been observed in nature and laboratory conditions between members of the Orthobunyavirus or Influenza viruses. The different factors which can lead to a reassortment are poorly understood. However disease outcome of reassortant virus infection may be very severe as illustrated by the haemorrhagic fever outbreak attributed to a reassortant orthobunyavirus in Africa and several pandemic outbreaks of influenza virus. One reason for the altered disease pattern of reassortant viruses might be a different interaction with cellular immune pathways leading to enhanced viral replication or increases immunopathology. Using reassortant orthobunyavirus we study the role of C-type lectin receptors for virus control and immunopathology (13, DFG-VIPER and Collaboration with Working Group Immunology). Within the project N-RENNT (VW Foundation and Lower Saxony ministry of science and education) we aimed to analyse the virus adaptation to different hosts within a natural infection cycle (Collaboration Institute for Pathology). We established infection models using ticks infected with TBEV and mice and virus from mouse and tick samples were subjected to sequencing analysis to see viral adaptation to vertebrate and invertebrates hosts.

iMINION

integrative Mücken-INterventIONsstrategien (iMINION)
Function in the Project    Project leader
Duration:    2020–2023
Funding bodies    BMBF German Research Platform for Zoonoses

 

Ansprechpartner: Prof. Dr. Stefanie Becker und Dr. Fanny Naccache, Institut für Parasitologie, Tierärztliche Hochschule Hannover
Weitere Projektpartner: PD. Dr. Michael Stern, Institut für Physiologie und Zellbiologie, Tierärztliche Hochschule Hannover und Dr. Kwang-Zin Lee, Arbeitsgruppe Insektenpathogene am Fraunhofer Institut für Molekularbiologie und angewandte Ökologie (IME)

 

 

 

 

 

Abstract:

Arboviral infections are caused by arboviruses which are transmitted by hematophagous arthropods, mainly mosquitoes and which repeatedly led to epidemic outbreaks in the last decades. In Germany, the most prevalent mosquito species with vectorial capacity is the indigenous Culex pipiens. For arboviral infections, vaccination is often not available and therefore vector control is crucial. The most common strategy for vector control is pesticide deployment, which can cause negative side effects for the ecosystem, and other pesticide exposed organisms. A combination of ISVs and a well-organized pesticide management can lead to effective vector control and protection of the ecosystem. Apart from arboviruses, mosquitoes are known to carry insect-specific viruses (ISVs). ISVs are described as non-pathogenic for vertebrates and have the potential to suppress arbovirus replication, which makes them more and more medically important. In this study, an interdisciplinary approach consisting of virological, behavioral and ecological perspectives is used to improve vector control measurements. The objective is to identify a suitable insect-specific virus and to establish a test system for different pesticides against mosquitoes. The newly established test systems will enable the investigation of the interaction of Cx. pipiens with insect-specific viruses and pesticides as well as the applicability of such a trilateral interaction in integrated mosquito control strategies examined. In general, two questions will be answered: 


1.    What role do insect-specific viruses play regarding the susceptibility of mosquitoes to pesticides?


2.    How can insect-specific viruses be combined with an optimized insecticide management for vector control and reduction of pesticides. 


Behavioral alterations impacted by virus infection through ISVs in combination with pesticide treatments will be investigated based on feeding experiments, electrophysiological measurements and activity assays.

ANTIGoNE

The role of ANTi-RVFV Immunity in GermliNe infection and bEhavior modulation in insects (ANTIGoNE)
Duration:    2019-2022
Funding bodies    DFG

 

Infection models and studies

 

Drosophila models: Using the well-characterised insect model Drosophila melanogaster, we were able to unravel several underlying immune mechanisms (7-11 and Figure 3). Within our project ANTIGONE (DFG) we will use this model and mosquito infection systems (7) to analyse the role of small RNA interference pathways on transovarial transmission and the influence of immune pathways on neuronal activity (Figure 4) and behavioural changes following an arbovirus infection. 

 

 

Figure 3: Intrinsic factors that interfere with the vector competence of mosquitoes.

The vector competence of a certain mosquito species is characterized by several factors. Firstly, the ability of the virus to overcome the midgut. Secondly the ability of the virus to replicate in various tissues of the insect host and most importantly the efficient dissemination of infectious viral particles to the saliva. The virus replication in the mosquito midgut is regulated by the gut microbiota represented by the Wolbachia endosymbiont (1) which can interfere with the virus replication by various ways. The virus replication in different tissues can trigger several antiviral pathways such as the RNAi pathways represented by Dicer cleavage and the inducible immune responses represented by the induction of Vago (2).

 

Figure 3 with figure legend were taken from: Schulz& Becker, Mosquitoes as arbovirus vectors: from species identification to vector competence. In: Mosquito-borne diseases: implications for public health. Parasitology Research Monographs, Band 10; Cham, Switzerland: Springer, 2018, 163-212.

 

 

Figure 4: Electroantennogram setup for Drosophila and mosquitoes to measure neuronal activity after different olfactory stimuli in infected and non-infected state.

 

Previous Research

Etablierung von Lektin-Bibliotheken aus Mensch, Schaf und Stechmücken – eine neue Plattform für Bindungsstudien mit viralen Glykoproteinen am Beispiel des Rifttalfiebers (GlycoViroLectinTool)
Duration:    2017-2018
Funding bodies    BMBF German Research Platform for Zoonoses

 

Niedersachsen-Research Network on Neuroinfectiology, (N-RENNT): Neuropathology of arthropod-borne Flavi- and Bunyaviruses
Duration:    2016-2018
Funding bodies    Niedersächsische Ministerium für Wissenschaft und Kultur

 

Dissecting the antiviral immune response in insects: Bunyaviruses as a model to unravel new roles of RNA silencing and persistence mechanisms
Duration:    2012-2015
Funding bodies    DFG

Bibliography (Selected publications)

1)    First Nationwide Surveillance of Culex pipiens Complex and Culex torrentium mosquitoes demonstrated the presence of Culex pipiens Biotype pipiens/molestus Hybrids in Germany.

Martin Rudolf, Christina Czajka, Jessica Börstler, Christian Melaun, Hanna Jöst, Heidrun von Thien, Marlis Badusche, Norbert Becker, Jonas Schmidt-Chanasit, Andreas Krüger, Egbert Tannich and Stefanie C. Becker

PLoS ONE (2013) DOI: 10.1371/journal.pone.0071832

 

2)    Distribution and genetic structure of Aedes japonicus japonicus populations (Diptera: Culicidae) in Germany.

Katrin Huber, Kathrin Schuldt, Martin Rudolf, Marco Marklewitz, Dina M Fonseca, Christian Kaufmann, Yoshio Tsuda, Sandra Junglen, Andreas Krüger, Norbert Becker, Egbert Tannich, Stefanie C. Becker

Parasitology research (2014) DOI: 10.1007/s00436-014-4000-z

 

3)    Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection.

Mayke Leggewie, Marlis Badusche, Martin Rudolf, Stephanie Jansen, Jessica Börstler, Ralf Krumkamp, Katrin Huber, Andreas Krüger, Jonas Schmidt-Chanasit,

            Egbert Tannich, Stefanie C. Becker.

One Health 2 (2016), DOI 0.1016/j.onehlt.2016.04.001.

 

4)    Aedes japonicus japonicus (Diptera: Culicidae) from Germany have vector competence for Japan encephalitis virus but are refractory to infection with West Nile virus.

Katrin Huber, Stephanie Jansen, Mayke Leggewie, Marlis Badusche, Jonas Schmidt-Chanasit, Norbert Becker, Egbert Tannich, Stefanie C. Becker

            Parasitology research 2014; DOI: 10.1007/s00436-014-3983-9

 

5)    Vector competence of field populations of the mosquito species Aedes japonicus japonicus and Culex pipiens from Switzerland for two West Nile virus strains.

Stefanie Wagner, Alexander Mathis, A.C. Schönenberger, Stefanie C. Becker, Jonas Schmidt-Chanasit, Cornelia Silaghi, Eva Veronesi

Med Vet Entomol. (2017), DOI: 10.1111/mve.12273

 

6)    First isolation and phylogenetic analyses of tick-borne encephalitis virus in Lower Saxony, Germany.

Mathias Boelke, Malena Bestehorn , Birgit Marchwald , Mareike Kubinski , Katrin Liebig , Julien    Glanz Claudia Schulz , Gerhard Dobler , Masyar Monazahian , Stefanie C. Becker Viruses (2019) DOI 10.3390/v11050462

 

7)    RNA interference restricts Rift Valley fever virus in multiple insect systems.

Isabelle Dietrich , Stephanie Jansen ,  Gamou Fall , Stephan Lorenzen , Martin

Rudolf , Katrin  Huber , Anna Heitmann ,Sabine Schicht , El Hadji Ndiaye, Mick Watson, Ilaria Castelli , Benjamin Brennan, Richard M. Elliott, Mawlouth Diallo,  Amadou A. Sall, Anna-Bella Failloux, Esther Schnettler, Alain Kohl, Stefanie C. Becker.  mSphere (2017) DOI 2:e00090-17

 

8)    Broad RNA Interference-Mediated Antiviral Immunity and Virus-Specific Inducible Responses in Drosophila.

Cordula Kemp*, Stefanie Müller*, Akira Goto, Vincent Barbier, Simona Paro, François Bonnay, Catherine Dostert, Laurent Troxler, Charles Hetru, Carine Meignin, Sébastien Pfeffer, Jules A Hoffmann and Jean-Luc Imler

The Journal of Immunology (2012) DOI: 10.4049/jimmunol.1102486;* equal contribution

 

9)    RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila.

Stefanie Müller, Valérie Gausson, Nicolas Vodovar, Safia Deddouche, Laurent Troxler, Jonathan Perot, Sébastien Pfeffer, Jules A Hoffmann, Maria-Carla Saleh and Jean-Luc Imler

Proceedings of the National Academy of Sciences (2010) DOI:10.1073/pnas.1014378107 1

 

10)  The DExD/H-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila.

Safia Deddouche, Nicolas Matt, Aidan Budd, Stefanie Müller, Cordula Kemp, Delphine Galiana-Arnoux, Catherine Dostert, Christophe Antoniewski, Jules A Hoffmann and Jean-Luc Imler

Nature Immunology (2008) DOI: 10.1038/ni.1664

 

11)  Dicing with viruses: microRNAs as antiviral factors.

Stefanie Müller and Jean-Luc Imler

Immunity (2007) DOI: 10.1016/j.immuni.2007.07.003 

 

12)  Culex torrentium mosquitoes from Germany are negative for Wolbachia.

Mayke Leggewie, Ralf Krumkamp, Marlis Badusche, Anna Heitmann, Stephanie Jansen, Jonas Schmidt-Chanasit, Egbert Tannich,  Stefanie C. Becker

Med Vet Entomol. (2017), DOI:10.1111/mve.12270

 

13)  The CARD9-associated C-type lectin, Mincle, recognizes La Crosse virus (LACV) but plays a limited

role in early antiviral responses against LACV

Joao T. Monteiro, Kathleen Schön, Tim Ebbecke, Ralph Goethe,Jürgen Ruland, Wolfgang Baumgärtner, Stefanie C. Becker, Bernd Lepenies 

Viruses (2019) DOI 10.3390/v11030303

Kontakt
Dr. Tina Basler
Tel.:+49 511 953-6141
Fax.:+49 511 953-8675
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