Title Promoter Affiliations Abstract "Aspects of porcine arterivirus entry in macrophages leading to virus persistence" "Hans Nauwynck" "Department of Translational Physiology, Infectiology and Public Health" "We want to investigate how entry of porcine arterivirus in macrophages via the macrophage-specific receptor sialoadhesin results in intracellular virus survival and virus persistence at cellular and animal level. An in vitro model will be set up to investigate how long virus remains infectious after sialoadhesin-dependent internatization in the macrophage." "Mechanistic study on virus entry and replication. A fundamental approach to the development of new and specific antiviral chemotherapeutics." "Jan Balzarini" "Laboratory of Virology and Chemotherapy (Rega Institute)" "Multitrofic interaction between tomato, plague and virus vector Frankliniella occidentalis, its natural enemy Orius laevigatus and tomato spotted wilt virus" "Patrick De Clercq" "Department of Crop protection" "The aim of this project is to research the way different insects (a herbivore and a natural enemy) and a pathogen (virus) influence each other and the host (tomato)." "Entry process of porcine arterivirus in macrophages and link with virulence/pathogenicity" "Hans Nauwynck" "Department of Translational Physiology, Infectiology and Public Health" "Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus in pigs that has a specific tropism for monocytic cells. The last two decennia, the virus is genetically changing, resulting in the infection of more and more subsets of monocytic cells. This causes higher virus replication and more disease. In the present project the basis of the monocyte switch will be examined." "Entry of feline coronaviruses in their primary target cells" "Hans Nauwynck" "Department of Translational Physiology, Infectiology and Public Health, Department of Biomedical molecular biology" "In this project the target cells of feline coronaviruses in intestinal mucosa will be identified and the replication in these cells will be studied in an intestinal mucosa explant model. Further, the entry of these viruses in the identified target cells will be analyzed and there will be searched for an explanation for the observed differences between FIPV and FECV." "Role of receptor tyrosine kinases and associated gangliosides in influenza virus replication" "Lieve Naesens" "Laboratory of Virology and Chemotherapy (Rega Institute)" "Influenza infections produce significant medical and socio-economic burden. Every year, human influenza A and B viruses cause millions of cases of severe respiratory illness and death. Every so often, a pandemic arises when a zoonotic influenza A virus with sustained human transmissibility enters the human population. Antivirals are crucial for influenza treatment and prevention, especially in fragile populations such as elderly. At the moment, neuraminidase (NA) inhibitors are the only drug class that is globally available, however resistance against these agents is a serious concern. New inhibitors with an entirely different mode of action are absolutely required. Besides targeting the virus itself, it is imperative to consider host cell factors that are less prone to resistance. Among the multitude of possibilities, we here focused on receptor tyrosine kinases (RTK), a class of protein kinases for which several therapeutics have been developed during the past decade.In Chapter 2, we first evaluated a library of 276 protein kinase inhibitors for anti-influenza virus activity in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 emerged as a robust inhibitor of influenza A and B virus replication, producing up to 3-log10 reduction in viral load at non-toxic concentrations. Detailed mechanistic investigations revealed that Ki8751 interferes with PDGFRβ-mediated influenza virus internalization, indicating that the virus matches with this specific RTK. By using two related CHO cell lines, we demonstrated that this route of virus uptake depends on gangliosides present on the cell membrane. Entering virus was shown to prefer GM3- over GM1-positive lipid rafts, consistent with a stimulating effect of GM3 on PDGFRβ signaling and virus uptake via this pathway. Our intriguing observation that the phosphorylated PDGFRβ undergoes desialylation by the viral NA, could indicate an as yet unknown function of NA in virus entry. PDGFRβ-mediated virus uptake coincided with activation of the Raf/MEK/Erk signaling pathway, but not of PI3K or phospholipase C-γ.In Chapter 3, we elaborated on the role of RTKs at later replicative stages, after observing that a VEGFR2-transfectant CHO cell line synthesized higher levels of viral RNA, an effect that was independent of cell surface-associated VEGFR2. The stimulating effect of VEGFR2 on viral RNA synthesis was strongest at 6 h p.i. and coincided with enhanced nuclear export of viral ribonucleoprotein (vRNP) complexes. We propose that nuclear VEGFR2 might alter the phosphorylation status of the viral nucleoprotein (NP), for instance at residues Tyr296/Ser297, and thereby prime the vRNPs for nuclear export. However, since the CHO cell line was found to express an incomplete form of VEGFR2 (i.e. lacking the kinase domain), the cooperation of another RTK appears required to alter the NP phosphorylation status and enhance influenza virus replication.In conclusion, our study indicates that cellular RTKs contribute to influenza virus replication at two stages: entry and nuclear export. To enter into the host cell, the virus efficiently exploits the PDGFRβ/GM3-mediated signaling pathway in which not only the viral HA but also its NA are playing a role. This tight association with PDGFRβ creates an opportunity to suppress virus replication with PDGFRβ inhibitors like Ki8751 or more advanced clinical candidates. To achieve robust activity against influenza A and B viruses, in combination with a favorable therapeutic window, inhaled formulations of PDGFRβ blockers deserve further attention. Besides, our findings indicate that viral nuclear export is regulated by RTK pathways acting in the nucleus, like VEGFR2-associated kinases or phosphatases. This plausibly involves altering the NP phosphorylation status. Since several RTK inhibitors are already approved or in clinical development, combining these therapeutics with direct acting antiviral drugs may be a valuable strategy to make influenza therapy more effective or address the antiviral drug resistance problem." "Study of the interactions between the Hepatitis C virus and the host ""in vitro"" en ""in vivo""" "This proposal aims at a better understanding of the interacties between the hepatitis C virus and the human host starting with ""viral entry"", early innate responses, the interferon signalling pathway, human Band T cell responses to structural and non-structural proteins of HCV. A variety of analytical tools will be used in addition to the chimeric mouse model for the study of HCV infection that has been developed recently by our research team." "Study of the earliest extracellular and intracellular interactions of the hepatitis C virus with human hepatocytes in chimeric uPA-SCID mice" "uPA-SCID mice can be infected with the hepatitis C virus (HCV) after they have been transplanted with human hepatocytes. In this research project we will evaluate in vivo the different steps in viral entry and evaluate different approaches to prevent HCV infection. In addition, we will map all changes in gene and protein expression that occur in the human hepatocytes that occur early after HCV entry. This may provide insight in the mechanism that HCV uses to evade the innate immune system of the human hepatocytes." "Characterisation of a novel Respiratory Syncytial Virus (RSV) immune evasion mechanism and evaluation of the impact for development of novel vaccines and prophylactic and/or therapeutic antibodies" "Peter Delputte" "Laboratory for Microbiology, Parasitology and Hygiene (LMPH)" "Respiratory Syncytial Virus (RSV) is the leading cause of severe lower respiratory disease in young children and is the second most important viral cause of respiratory disease in adulthood, after influenza virus. Currently, no vaccines and no antivirals are available to control RSV infections. Recent advances in RSV research have shown that the F protein is a major target for development of novel therapy and prophylaxis. F-specific antibodies, such as Palivizumab, are available, but were shown not to be fully effective, since hospitalization rates are reduced only by 55%. In addition, vaccines may also not fully protect against infection. This is exemplified by the fact that even after natural infection(s) and induction of F-specific neutralizing antibodies, RSV is able to reinfect. Since re-infection occurs without considerable antigenic change, there must be a different immune evasive mechanism than that influenza A virus reinfections, which is dependent on antigenic drift/shift.Our understanding of RSV F protein functionality, besides its role in fusion, is however insufficient to explain the immune evasive mechanisms involved. Currently there is a great risk that newly developed monoclonal antibodies and vaccines will suffer from constrained effectiveness.Very recently, preliminary studies in our research group have shown that upon binding of RSV-specific antibodies to RSV F protein expressed on the surface of infected cells, internalization occurs of RSV protein-antibody complexes. As a result, infected cells will most likely no longer be efficiently detected and eliminated by antibody-based immunity. In addition, a reduction in the expression of RSV proteins on the surface of infected cells may interfere with the immunogenicity and hamper the induction of strong immune responses. This novel finding may have profound effects on (1) our understanding of RSV pathogenesis and the occurrence of frequent RSV reinfections and (2) the development of new RSV vaccines and monoclonal antibodies. It is therefore the aim of this project to fully characterize this endocytosis process up to the molecular level, both in vitro and in vivo. We will (A) characterize the endocytic process and the consequences for RSV immune evasion, (B) identify amino acids of the F protein involved and create F proteins that show no internalization, (C) generate recombinant viruses with mutant F proteins lacking endocytic properties and (D) evaluate the recombinant viruses in vivo." "Vaginal lactobacilli in the prevention of HSV-2 infection." "Jos Vanderleyden" "Centre of Microbial and Plant Genetics" "Humans live in symbiosis with a tremendous number of bacteria, collectively referred to as the microbiota, that play a key role in several host physiological processes and health. While the gut microbiota has received plenty of attention the past decades, the vaginal microbiota is only recently gaining interest as a crucial player in female and reproductive health. The vaginal microbiota of most healthy women is generally dominated by Lactobacillus species, recognized as a biomarker species for vaginal health. These species also play an indispensable role in supporting the host’s defence against a wide variety of bacterial, fungal and viral pathogens. However, a detailed molecular understanding of their adaptation to the vaginal niche and their health promoting and anti-pathogen effects is currently lacking. The goal of this PhD project is therefore to deliver insights on the molecular mechanisms used by lactobacilli to contribute to vaginal health, and to defend against herpes simplex virus type 2 (HSV-2), the main causative agent of genital herpes disease, as a case study viral pathogen in the vaginal tract.Residing at the port of entry of various pathogens causing urogenital and sexually transmitted infections in women, lactobacilli could promote vaginal health by enforcing the vaginal barrier function against these pathogens. As the vaginal barrier is composed of the vaginal epithelium, mucus and the immune system, this PhD research investigated interactions of lactobacilli with the host such as adhesion and immunomodulation as key properties to a healthy vaginal environment. Ultimately, this knowledge will drive more targeted selection criteria for probiotic strains focused on the maintenance/restoration of vaginal health.Given that a vaginal microbiota not dominated by lactobacilli has been identified as a risk factor for HSV-2 infections, a second part of this research focused on the elucidation of putative antiviral mechanisms of action of lactobacilli against HSV-2. Hereto, a platform to study interactions between bacteria, cells and viruses was developed, representing a significant technical challenge of this project. Three antiviral mechanisms were investigated: (1) co-aggregation with virions thereby preventing host cell invasion, (2) competition with attachment/entry receptors of HSV-2 thereby blocking virus adhesion, and (3) stimulation of the innate antiviral immune response of vaginal epithelial cells following viral challenge. Next to delivering pioneer work on Lactobacillus-virus interactions at the molecular level, this PhD findings open new perspectives for microbial management-based prevention and treatment strategies against HSV-2 and potentially other vaginal infections."