One Health EJP PhD Grants First Round Current state of the art in the chosen area and the knowledge gap being addressed In vitro models for risk assessment of disease and transmission of emerging zoonotic viruses. Following detection of an emerging zoonotic pathogen, there is a pressing need to know some essential characteristics in order to assess the risk to human health: 1) is it capable of infecting humans? 2) can it spread between humans? 3) Can it cause (severe) disease in humans? Acquisition of efficient replication and transmission capabilities are key determinants of emerging zoonotic strain adaptation to the human host; these distinct properties determine what the potential public health impact of a rare zoonotic event can be and therefore are a hallmark of emerging infectious disease preparedness, but may be challenging to assess. Risk assessments typically are based on observational studies during outbreaks, and it has been challenging to obtain reliable comparative information on virus properties that impact on their ability to spread among humans. Animal models are widely used to study viral replication kinetics, virus-host interactions, transmission and pathogenesis, however, the success rate of extrapolation to humans of these results is highly dependent on the pathogens that are being studied, the animal models that are being used and the research question at hand. Therefore, there is an urgent need for clinically relevant, and rapidly deployable in vitro models of human and animal organ systems to obtain reliable information on virus properties that impact on their ability to spread to and among humans and on their pathogenicity. Our underlying rationale is that important properties of such emerging pathogens can be explored by use of well characterised in vitro cell and tissue systems that mimic the natural route of infection and primary target organ systems in the animal hosts and humans. Key questions for which cell- and tissue culture models are used address host range, cell and tissue tropism in relation to the potential for shedding in reservoir hosts and humans. Furthermore, cell culture systems are the first basis for studying changes of the transcriptome pattern in infected in comparison to control cells, which gives insights into possible cellular mechanisms affected or circumvented by the pathogen, providing clues to the observed pathology and disease. For this project we will use a panel of related viruses with known zoonotic potential, and either resulting in (severe) disease, asymptomatic infection or unknown infection in humans. We will focus on the order of Bunyavirales (orthohantavirus, Schmallenbergvirus, Oropouche virus, Rift Valley fever virus, Shuni virus). First, viruses from this order include orthohantaviruses (such as Dobrava, Seoul and Sin Nombre viruses) and phleboviruses (Rift valley fever virus ; RVFV) which are both listed in the top 10 of the Emerging Zoonoses Information and Priority system of the Dutch National Institute for Public Health and the Environment (RIVM). In addition, RVFV is on the WHO list of Blueprint priority diseases. Finally, we propose to study orthobunyaviruses as the non-zoonotic Schmallenberg virus (SBV) in comparison to the related zoonotic Oropouche viruses and the further spreading Shuni virus. These viruses would provide a unique model of related species with different reservoirs and a distinct zoonotic risk. Testable hypothesis and proposed methods

Our central hypothesis is that the ability of emerging animal viruses to transmit to humans and cause disease, can be predicted using in vitro models of human and animal organ systems. We plan to test our central hypothesis by pursuing the following three specific aims: Aim 1. Determine the ability of animal viruses to infect humans: in vitro models will be used that represent crucial steps in the emergence of zoonotic viruses, including cell types predicting susceptibility and host range as well as the portal of entry and dissemination (respiratory, intestinal, and endothelium). In vitro models will include primary cells as well as precision cut organ slices of primary target organs such as lung, kidney (orthohantavirus) and liver (RVFV; orthobunyaviruses). Representative models will be compared between animal and human hosts in parallel. We will determine: a) virus replication efficiency; b) cell tropism; c) receptor expression (if known) and d) sites of virus entry and release. By modelling this crucial first step in zoonotic transmission we can identify key determinants for susceptibility to infection.

One Health EJP: Application form for PhD project call 1 Aim 2. Determine the potential of emerging zoonotic viruses to transmit to humans via the respiratory route: many of the emerging zoonotic viruses can enter the human host through the respiratory tract, however the barriers for respiratory transmission are unknown for many viruses. We will use primary human nasal and tracheobronchial ciliated epithelial cells as models of proximal airway epithelium and cultures of alveolar epithelial cells as models of distal lung regions. Cells will be grown on filters and differentiated under air-liquid interface (ALI) conditions to generate a polarized pseudo- stratified epithelium that recapitulates the morphological features of the human airways. These ALI will be cultured at 34°C or 37°C to accurately mimic and control for the temperature differences between upper and lower respiratory tract. We will infect them with orthohantaviruses which are known to enter the human host via inhalation and compare this with infection with RVFV or orthobunyaviruses for which respiratory transmission in humans has not been reported. We will: a) determine the cell tropism; b) sites of virus entry and release and c) cytopathic and functional effects on the epithelial cells. These will include co-staining of viral antigen and cell type specific markers, staining for apoptosis markers and quantifying epithelial cell functions such as barrier integrity and mucus production. By using well characterized in vitro models of the human respiratory tract we will be able to compare the ability of zoonotic viruses to infect the respiratory tract as a potential indicator for respiratory transmission. Aim 3. Determine the potential of zoonotic viruses to cause disease in humans: the ability of a virus to cause disease depends in a large part on the effect of virus infection on the host (induction of host response) as well as the effect of that host response on the virus and host itself. Virus infections generate a specific pattern of changes by genome-wide interactions with the host. We propose to evaluate the complete transcriptome of the primary target cells in animal and human hosts to enable the identification of common genes that are upregulated or downregulated upon viral infection in the absence of disease (orthohantaviruses in rodents; orthobunyaviruses in adult livestock, SBV in humans) or presence of disease (orthohantavirus infection in humans, and RVFV infection in animals and humans; Oropouche virus in humans). Specific transcriptome patterns will be defined by deep RNA sequencing (RNAseq) and microarray analyses will be used for genome-wide screening of infected and non-infected cells; this high-resolution analysis will allow the identification of differentially expressed host genes. Pathways of interest, may be confirmed by targeting for knock-out or knock-down experiments using CRISPR/Cas9 mediated site- directed-mutagenesis. In addition to the methods above, which evaluate the host response of a population of cells, we will use techniques to visualize individual (m)RNA molecules inside a single cell. These so-called single molecule fluorescence in situ hybridization technique (RNA-FISH) can be used to evaluate mRNA responses in in vitro models. These novel methods will allow us to identify key molecular mechanisms of pathogenesis common for these zoonotic viruses as potential biomarkers for disease development and severity. Relevance of PhD topic to One Health EJP strategic research agenda and overarching objectives of the EJP This PhD topic fits well within the EJP research topic domain Emerging Threats, with the objective to identify host tropism, virus transmission, and virus-host interactions in a One Health approach, and an emphasis on applicability of rapid laboratory-based hazard characterisation to provide input for risk assessments using state-of-the-art in vitro systems and analysis tools. This proposal aims to create a network of interdisciplinary collaborations to develop a model for emerging zoonotic virus characterization using state-of-the-art in vitro systems and analysis tools. Principle and collaborative supervisor’s experience and suitability Principle supervisor (co-promoter): Dr. Barry Rockx, is head of the Exotic Viruses workgroup at the Department of Viroscience at Erasmus MC (1fte). EMC is a partner in the Netherlands Centre for One Health (NCOH), which is a formal collaboration between academic groups and public health institutes to address One Health challenges. Dr. Rockx has over 10 years expertise in working with emerging zoonotic viruses causing hemorrhagic, respiratory and neurological diseases, including arboviruses, orthohantaviruses, henipaviruses and filoviruses in a variety of in vitro and in vivo models.

One Health EJP: Application form for PhD project call 2 Promoter: Prof. Dr. Marion Koopmans is head of the Department of Viroscience at Erasmus Medical Centre. She is also directing one of the four research pillars, the Emerging Infectious Disease theme, in the NCOH. Her research interests are in understanding virus ecology, epidemiology and evolution. Second promotor: Prof. Dr. Jeroen Kortekaas is the head of the Arboviral Zoonoses group at Wageningen Bioveterinary Research (WBVR) and Extraordinary professor of Veterinary Arbovirology at Wageningen University. The main responsibility of his team is to develop vaccines for the control of notifiable transboundary arboviral diseases. He has a key expertise on Rift Valley fever virus (RVFV) and other members of the family Bunyaviridae. Collaborative supervisor: Prof. Dr. Martin Beer is the Director of the Institute of Diagnostic Virology of the Friedrich-Loeffler-Institut (FLI), and Extraordinary professor at the Ludwig-Maximilians-University Munich. He has expertise in working with different animal viruses, including Bunyaviridae, with special emphasis on diagnostics, genetic characterization, phylogeny, and in vivo characterization. Institutional PhD student support The PhD student will be positioned within the NCOH. The PhD student will be embedded in and supported by the Department of Viroscience of Erasmus Medical centre, which is a National and EU reference laboratory for emerging viral diseases and WHO collaborating centre. The PhD candidate will be co-supervised and seconded to work on models of orthobunyavirus infection (WBVR) and characterization of the host gene expression (FLI), to ensure a multidisciplinary approach and enhance the training of the next generation of talented translational researchers by bringing together top-level expertise on virology, molecular biology and cell biology to address urgent public health questions in the field of emerging infectious diseases. Opportunities for networking in addition to existing and planned EJP activities (International and Interdisciplinary (med-vet-environment)) This project provides key infrastructural developments within an international and interdisciplinary med-vet-environment for improving overall global One Health responses against viruses as part of several projects coordinated by researchers at the Erasmus MC, WBVR and FLI, including EVD-LabNet, EMERGE, PREPARE, COMPARE and ZAPI, whose overall aims are to share virological, epidemiological, molecular data on and pipelines for therapeutic development for emerging viruses, through international networks of public health institutes, veterinary institutes and universities. The expertise that is being developed within these projects will greatly facilitate this envisioned EJP PhD project. Title of linked EJP Joint Research Project or Joint Integrative Project (if applicable) Not applicable Budget and Details of co-funding (either confirmed or proposed) The total budget of the 4-year project: € 206.964 PhD student (1 fte, 4 years) € 127.448 Supervision (0,2 fte, 4 years) € 80.000 Consumables (4 years x 20.000 euro) € 4.000 Conferences (4 meeting, 1.000 euro per meeting) € 3.000 Travel (travel costs for secondments at WBVR and FLI) € 5.000 publication cost (4 publications, 1.250 per publication) € 426.412 total direct cost € 106.603 25% indirect cost € 533.015 total budgeted cost

Co-funding for the salary, consumables, travel and publication costs of the Ph.D. student and co-funding for supervision is confirmed by the Department of Viroscience, Erasmus MC. Please return the completed form to ([email protected]) by 16:00 CET 20th April 2018

One Health EJP: Application form for PhD project call 3