Research priority focus (RPF) HOST-­‐PATHOGEN INTERACTIONS Vetsuisse Faculty, University of Bern Principal investigators, research topics & contacts October 2015 Marco Alves, Institute of Virology and Immunology marco.alves@vetsuisse.unibe.ch Research interests Role of mesenchymal stem cells in respiratory virus pathogenesis. We aim at dissecting the role of mesenchymal stromal cells (MSCs) in respiratory virus pathogenesis. MSCs are non-­‐hematopoietic stem cells, which were originally described in the bone marrow cavity, and possess mesenchymal regenerative capacities. They have recently been found in a number of organs, including the lung, and have the capacity to differentiate into osteocytes, chondrocytes and adipocytes. Moreover, MSCs cultivated in airway-­‐specific growth media are able to differentiate into airway epithelial cells (AECs) such as type I alveolar cells, and Clara cells. Because of this large panel of differentiation capability, MSCs are believed to play an important role in the lung epithelium homeostasis and repair. MSCs have been described as organ-­‐resident progenitor cells, and may play a key role in support of local progenitor cells, such as bronchoalveolar stem cells, creating a hematopoietic stem cell-­‐like niche. Resident MSCs are considered similar to pericytes and have been located in perivascular location of most organs in close contact with capillaries and blood cells. In addition, MSCs are endowed with a repertoire of cell surface receptors that enables them to sense viruses and their byproducts and because of this may be critically involved in modulating the host immune response to invading pathogens in order to maintain tissue homeostasis. Main collaborations •
Prof. Artur Summerfield, University of Bern, Switzerland •
Dr. Sean Hall, University Hospital of Bern, Switzerland •
PD Dr. Alexander Möller, University Hospital of Zürich, Switzerland •
Prof. Laurent Kaiser, University Hospital of Geneva, Switzerland Selected publications • Schögler A, Muster RJ, Kieninger E, Casaulta C, Jung A, Möller A, Geiser T, Regamey N, and Alves MP. Vitamin D represses rhinovirus replication in cystic fibrosis cells by inducing LL-­‐37. Eur Respir J, 2015 (in press) • Alves MP, Schögler A, Ebener S, Vielle NJ, Casaulta C, Jung A, Moeller A, Geiser T, and Regamey N. Comparison of innate immune responses towards rhinovirus infection of primary nasal and bronchial epithelial cells. Respirology, 2015 (in press) • Schögler A, Kopf BS, Edwards MR, Johnston SL, Casaulta C, Kieninger E, Jung A, Möller A, Geiser T, Regamey N, and Alves MP. Novel antiviral properties of azithromycin in cystic fibrosis airway epithelial cells. Eur Respir J, 2014 • Tang AC, Turvey SE, Alves MP, Regamey N, Tümmler B, and Hartl, D. Current concepts: host-­‐pathogen interactions in cystic fibrosis lung disease. Eur Respir Rev, 2014. • Kieninger E, Singer F, Tapparel C, Alves MP, Latzin P, Casaulta C, Tan HL, Bossley C, Bush A, Davies JC, Kaiser L, and Regamey N. High rhinovirus burden in lower airways of children with cystic fibrosis. Chest, 2013. Rupert M. BRUCKMAIER, Veterinary Physiology, University of Bern rupert.bruckmaier@vetsuisse.unibe.ch Research interests Mastitis and the mammary gland immune system of the dairy cow: Studies are performed both in vivo and in vitro with primary mammary epithelial cell cultures. We have been studying the immune response to different bacterial components such as LPS and LTA (as models for mastitis caused gram-­‐negative vs. gram-­‐positive bacteria. Recently we have focused, beyond the innate immune response, also on the activation of the adaptive immune system via the transfer of immunoglobulins from blood into the mammary gland. In parallel we are also interested in the mechanisms of colostrum formation which represents another event that involves the transfer of immunoglobulins from blood into milk, albeit another immunoglobulin subtype (IgG2 vs. IgG1). Besides the local effect at the level of the mammary gland we are also interested in influence of the metabolism mainly in transition cows on the immune response, such as the inhibitory effect on the immune system by elevated ketone body plasma concentrations which influence the secretion of glucagon from the pancreas and hence the availability of glucose as an energy fuel for the immune system. Currently we work on the potential to manipulate the blood-­‐milk barrier (tight junctions etc.) in mastitis cases where the adaptive immune response is not activated, which seems to be mostly the case in response to pathogens that typically cause chronic mastitis. Such a treatment could be of special interest in vaccinated animals where the specific antibodies may be present in the blood but are not transferred into the milk. Funding: SNF; DFG; industry; private foundations Main collaborations Roger Stephan (Vetsuisse Zürich), Joachim Frey (Vetsuisse Bern): Isolation of different bacterial strains for in vivo and in vitro experiments Techniques In vivo experiments with dairy cows, biopsy techniques from mammary gland, liver and other organs; primary mammary cell cultures including transwell technique, quantitative PCR; ELISA, RIA for various cytokines, growth factors and hormones, radio-­‐
receptor-­‐assays, various staining techniques for biopsy samples, electron microscopy More information under http://www.vetphysio.unibe.ch 5 recent publications •
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O. Wellnitz, E.T. Arnold, R.M. Bruckmaier: Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland. J. Dairy Sci. 94 5405-­‐5412 2011 O. Wellnitz, E.T. Arnold, M. Lehmann, R.M. Bruckmaier: Short communication: Differential immunoglobulin transfer during mastitis challenge by pathogen-­‐specific components. J. Dairy Sci. 96 1681-­‐1684 2013 M. Zarrin, O. Wellnitz, H.A. van Dorland, R.M. Bruckmaier: Induced hyperketonemia affects the mammary immune response during lipopolysaccharide challenge in dairy cows. J. Dairy Sci. 97 330-­‐
339 2014 C. Zbinden, R. Stephan, S. Johler, N. Borel, J. Bünter, R.M. Bruckmaier, O. Wellnitz: The inflammatory response of primary bovine mammary epithelial cells to Staphylococcus aureus strains is linked to the bacterial phenotype. PLoS One Jan. 30; 9 (1) 2014 C.R. Baumrucker, R.M. Bruckmaier: Colostrogenesis: IgG1 Transcytosis Mechanisms. J. Mammary Gland Biol. Neoplasia 19 103-­‐117 2014 Dominik BURGER, Institut suisse de médecine equine (ISME), University of Bern and Agroscope dominik.burger@vetsuisse.unibe.ch Research interest MHC and reproductive strategies in horses: The reproductive efficiency of domesticated horses is often lower than what could be expected from observations in feral herds. In the wild, stallions typically live with mares in harem bands, with other stallions in bachelor bands, or occasionally in mixed sex transitional bands. Hereby foaling rates of up to 90% are achieved, compared to approximately 75% in well-­‐
managed domesticated horse populations. An interdisciplinary view and analysis of the intra-­‐ and intersexual interactions between stallions and mares and their effects provide new potentially important information, which in the future may be used for optimization of breeding management. For that reason new biotechnological methods are currently established in the ISME in Avenches and Bern, which are in the interest of future scientific and practical breeding purposes. A main focus in this context is the influence of the major histocompatibility complex (MHC). The MHC does not only play a crucial role in the adaptive immune response of vertebrates, but has also an important role in social signalling, from parent-­‐progeny and other kinds of kin recognition to mate choice and further contexts of inter-­‐sexual communication. Findings in our studies suggest that the MHC or linked genes may influence mate choice in horses in a similar manner to previous observations in human beings and other species, and that the horse is a promising model for future research on MHC-­‐linked social signalling. Actually we perform experiments in order to increase the present knowledge of female and male reproductive strategies during the time of fertilization in mammals, leading to higher fertility and biodiversity. This research could help us to better understand the significance of sexual selection and life-­‐history decisions at the level of gamete fusion and early pregnancy, in order to reduce inbreeding and/ or to increase MHC heterozygosity and improve resistance to infections. Funding: Agroscope; ISMEquine Research Main collaborations Eliane Marti, Tosso Leeb, Rupert Bruckmaier (Bern) Claus Wedekind (Lausanne), Ghylene Goudet (F-­‐Nouzilly), Harald Sieme (D-­‐Hannover) Techniques Equine leucocyte antigens (ELA) serotyping, microsatellite PCR, artificial insemination, embryo collection and puncture, in vitro fertilization, in vitro ovum pick-­‐up, in vivo ovum pick-­‐up, in vitro fertilization (IVF) and intra-­‐cytoplasmatic sperm injection (ICSI) More information under http://ismequine.ch/wp/?page_id=371&lang=en Vince GERBER, Institut suisse de médecine equine (ISME), University of Bern and Agroscope vinzenz.gerber@vetsuisse.unibe.ch Research interests Genetics of equine recurrent airway obstruction: Allergies and hypersensitivity disorders are not only increasingly affecting humans, but also companion animals. The “hygiene-­‐hypothesis” postulates an inverse relationship between certain parasitic and other infectious diseases and hypersensitivties as a result of an interaction of genetic and environmental factors. Recurrent airway obstruction (RAO; horse asthma), a major health problem of stabled horses, is a severe, debilitating disease, characterized by coughing and increased breathing effort due to cholinergic bronchospasm and airway hyperreactivity as well as neutrophil and mucus accumulation in the airways as a result of hypersensitivity reactions to inhaled allergens. Our study population consists of two large half-­‐sibling families (>100 offspring per sire) and large numbers of unrelated RAO cases and controls (>600). We identified regions on chromosomes 13 and 15 as well as specific cytokine (e.g. Th2-­‐type, such as IL4R and IL13) and chemokine (e.g. CXCL13) gene expression profiles associated with RAO. Strongylid egg shedding was associated with regions on chromosomes 15 and 20 and with Th2-­‐type (e. g. IL4R) and other cytokine profiles. These results differed between the families, indicating genetic heterogeneity. Intriguingly, in one of the families and in unrelated RAO-­‐affected horses, RAO is strongly associated with other, in some horses with multiple hypersensitivities, specifically concerning the skin, and with resistance to parasites. Funding: SNF; Agroscope; ISMequine Research Main collaborations Tosso Leeb, Gaudenz Dolf, Eliane Marti, Bruno Gottstein and Caroline Frey (Bern) Stefan Rieder, Marcus Neuditschko (Agroscope) J-­‐P. Lavoie (Montreal, Ca) Techniques SNP genotyping; RNAseq Transcriptome analyses; Clinical phenotyping, parasites egg counts; blood mononuclear cell stimulation; quantitative PCR More information under http://ismequine.ch/wp/?page_id=1135&lang=en Bruno GOTTSTEIN, Institute of Parasitology bruno.gottstein@vetsuisse.unibe.ch Host-­‐parasite interactions during infection with the cestode parasite Echinococcus multilocularis: humans represent an aberrant intermediate host for the fox tapeworm Echinococcus multilocularis, while small rodents (mice) function as natural intermediate hosts. Infection in humans can cause alveolar echinococcosis (AE), a severe disease with fatal outcome if not treated appropriately. We have previously shown that E. multilocularis infection affected peritoneal DCs such as to remain in an immature or resting state as well, characterized by low expression of costimulatory molecules and MHC class II (I-­‐a) molecules. A concomitant high level of TGF-­‐β-­‐expression classifies these DCs within cells with suppressive features. Together, impairment of MØ and DC maturation and antigen presentation indicated that a successful E. multilocularis infection induces an anergic status of immunity in the host. Subsequent experiments at the T cell level documented that regulatory T cells (Tregs) interfere in the complex immunological host response to infection. Indeed, a subpopulation of regulatory CD4+CD25+ T cells isolated from E. multilocularis-­‐infected mice reduced ConA-­‐driven proliferation of CD4+ T cells. The high expression levels of Foxp3 mRNA by CD4+ and CD8+ T cells suggested that subpopulations of regulatory CD4+Foxp3+ and CD8+Foxp3+ T cells were involved in negatively modulating the immune responses of E. multilocularis-­‐infected mice. Our most recent data demonstrated now that a novel CD4+CD25+ Treg effector molecule FGL2 contributes to the outcome of E. multilocularis infection by promoting Treg cell functions; they give evidence for a role of IL-­‐17 in FGL2 regulation, and suggest that targeting FGL2 could be used for the development of novel treatment approaches in this parasitic disease. Further areas of research are: (i) development of in vitro culture system E. multilocularis primary cells; (ii) generation of recombinant single chain anti-­‐E. mulitlocularis antibodies expressed in E. coli based on the monoclonal antibody anti-­‐Em2/G11, tob e used for diagnostic and therapeutic purposes; (iii) molecular and immunological characterization of the P29 antigen in E. granulosus and E. multilocularis Funding: Swiss National Science Foundation Main collaborations M. Siles-­‐Lucas, Salamanca D. Vuitton, Dr. L. Millon, F. Grenouillet, O. Blagosklonov, J. Knapp, Besançon P. Deplazes, P. Torgerson, Zürich; B. Mühllhaupt, Zürich; K. Brehm, Würzburg Selected recent publications: •
Boubaker B, Hemphill A, Huber CO, Spiliotis M, Babba H, Gottstein B: Prevention and immunotherapy of secondary murine alveolar echinococcosis employing recombinant EmP29 antigen. Plos Negl Trop Dis. 9(6):e0003795 (2015). •
Wang J, Vuitton DA, Hemphill A, Müller N, Blagosklonov O, Grandgirad D, Leib S, Shalev I, Levy G, Lu X, Lin R, Wen H, Spiliotis M, Gottstein B: Deletion of fibrinogen-­‐like protein 2 (FGL-­‐2), a novel CD4+ CD25+ Treg effector molecule, leads to improved control of Echinococcus mutilocularis infection. PLoS Negl Trop Dis. 9(5):e0003755 (2015) •
Gottstein B, Wang J, Boubaker G, Marinova I, Spiliotis M, Müller N, Hemphill A. Susceptibility versus resistance in alveolar echinococcosis (larval infection with Echinococcus multilocularis). Vet Parasitol. pii: S0304-­‐4017(15)00369-­‐6 (2015). •
Gottstein B, Stojkovic M, Vuitton DA, Millon L, Marcinkute A, Deplazes P. Threat of alveolar echinococcosis to public health -­‐ a challenge for Europe. Trends Parasitol. (9):407-­‐12 (2015). •
Wang J, Lin R, Zhang W, Li L, Gottstein B, Blagosklonov O, Lü G, Zhang C, Lu X, Vuitton DA, Wen H. Transcriptional profiles of cytokine/chemokine factors of immune cell-­‐homing to the parasitic lesions: a comprehensive one-­‐year course study in the liver of E. multilocularis-­‐
infected mice. PLoS One, 9(3):e91638 (2014). Andrew HEMPHILL, Institute of Parasitology andrew.hemphill@vetsuisse.unibe.ch Research interests Development of (immuno-­‐)therapeutical options that lead to prevention and treatment of protozoan and helminth infections. More specifically, protozoan and helminth models are investigated: (i) the apicomplexan parasites Neospora caninum, Toxoplasma gondii, Theileria annulata and Besnoitia besnoiti, and (ii) the cestode Echinococcus multilocularis, the causative agent of alveolar echinococcosis in humans. For Neospora caninum, proteins functionally involved in host cell entry are promising vaccine candidates, and a number of recombinant recombinant and chimeric vaccines have been characterized, which limit parasite dissemination and fetal infection in experimentally infected mice. In addition, we have studied the in vitro and in vivo effects of novel candidate compounds and identified drug-­‐interaction partners in parasite and host cells, some of which also are successfully targeted by vaccines. More recently, we have focussed on inhibitors of calcium-­‐dependent protein kinases (CDPKs), inhibit host cell invasion and also egress. A combined approach employing vaccination and chemotherapeutical treatment is being envisaged as a potential option to limit the effects of these parasitic infections. The current treatment options for echinococcosis are very limited. A metacestode culture system and parasite stem cell cultivation has been established, and in combination with the mouse model these options are being exploited for identifying novel compounds. Thiazolides, dicationic compounds, ruthenium-­‐based metallo-­‐organic drugs, proteasome inhibitors, mefloquine, artemisinins and other anti-­‐malarials have been investigated so far, some of which with promising in vitro and in vivo activity. Using affinity-­‐chromatography with epoxy-­‐
activated sepharose-­‐linked compounds we identified several drug-­‐binding proteins that are involved in the mechanisms of action of these compounds. Funding: SNF; SBFI; NIH/USDA; several private foundations Main collaborations Adrian Hehl (Zürich) & Alexandre Leitao (Lisbon): Genome and transcriptome of Besnoitia besnoiti Alexandre Leitao and Afonso Basto (Lisbon): OprI-­‐mediated immunomodulation Wes VanVoorhis (Seattle): CDPK-­‐inhibitors in apicomplexan parasites Luis Ortega-­‐Mora (Madrid): N. caninum infections and vaccine/drug efficacy in sheep. Norbert Müller, Joachim Müller (Bern): recombinant protein expression and purification Julien Furrer (Dept of Chemistry, Bern): ruthenium-­‐based drugs Techniques Culture of parasites and mammalian cells; immunofluorescence; electron microscopy; mouse infection experiments and vaccine/drug trials; recombinant protein expression; quantitative PCR; basic immunological techniques 5 selected publications •
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Küster T, Stadelmann B, Rufener R, Risch C, Müller J, Hemphill A. Oral treatments of Echinococcus multilocularis-­‐infected mice with the antimalarial drug mefloquine that potentially interacts with parasite ferritin and cystatin. Int J Antimicrob Agents. 2015 Sep 2. [Epub ahead of print] PubMed PMID: 26395219. Winzer P, Müller J, Aguado-­‐Martínez A, Rahman M, Balmer V, Manser V, Ortega-­‐Mora LM, Ojo KK, •
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Fan E, Maly DJ, Van Voorhis WC, Hemphill A. In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-­‐Forming Apicomplexans Toxoplasma gondii and Neospora caninum. Antimicrob Agents Chemother. 2015 Oct;59(10):6361-­‐74. Müller J, Aguado-­‐Martinez A, Manser V, Balmer V, Winzer P, Ritler D, Hostettler I, Arranz-­‐Solís D, Ortega-­‐Mora L, Hemphill A. Buparvaquone is active against Neospora caninum in vitro and in experimentally infected mice. Int J Parasitol Drugs Drug Resist. 2015 Feb 13;5(1):16-­‐25. Arranz-­‐Solís D, Benavides J, Regidor-­‐Cerrillo J, Fuertes M, Ferre I, Ferreras Mdel C, Collantes-­‐
Fernández E, Hemphill A, Pérez V, Ortega-­‐Mora LM. Influence of the gestational stage on the clinical course, lesional development and parasite distribution in experimental ovine neosporosis. Vet Res. 2015 Mar 3;46:19 Stadelmann B, Aeschbacher D, Huber C, Spiliotis M, Müller J, Hemphill A. Profound activity of the anti-­‐cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes. PLoS Negl Trop Dis. 2014 Dec 4;8(12):e3352. More information under http://www.vetsuisse.unibe.ch/ipa/content/index_ger.html Christoph KOCH, Institut suisse de médecine equine (ISME), University of Bern and Agroscope christoph.koch@vetsuisse.unibe.ch Research interest Equine Sarcoid Disease (host-­‐pathogen-­‐interactions, genetic background, immunology, papilloma virus as pathogen): The equine sarcoid (ES) is by far the most common skin tumor found in equids. Sarcoids have a high capacity for local tissue invasion of the dermis and subcutis but true metastatic dissemination does not occur. Although the disease is rarely fatal, tumors may become ulcerated or infected, and recrudescence is frequently observed after tumor removal. Therefore, equine welfare issues and economic aspects must be considered when evaluating and treating this disease. Bovine papilloma virus (BPV-­‐1/-­‐2) is regarded as an essential extrinsic factor in the development of ES, and transmission of BPV is supposed to occur from cattle to horses or from horse to horse. However, intrinsic factors in particular a genetic predisposition, is also thought to be an important determinant in the clinical manifestation of ES disease. Initially, family predispositions and later breed predilections for disease were observed, suggesting a genetic basis to an individual’s susceptibility to sarcoids. These suppositions were substantiated when associations of ES with certain equine leukocyte antigen alleles were found. In recently published segregation analysis of FM horses affected with sarcoid disease, we found that variation in susceptibility to ES is best explained by a model incorporating polygenic variation, with an estimated heritability of 21%. Recent studies suggest that regulatory T cells (Tregs) are associated with disease severity and progression in papilloma virus induced neoplasia. The combined aim of several current projects is to investigate the role of Tregs in the host-­‐pathogen interaction of BPV-­‐induced equine sarcoid disease. We have demonstrated an increased expression of Treg markers (FOXP3) in tumour-­‐associated tissues of ES-­‐affected equids compared to biopsies from areas of unaffected skin and skin of healthy subjects. This indicates a local, Treg-­‐induced immune suppression. Furthermore, we could show that tumor-­‐associated lymphocyte infiltrates (cytotoxic T-­‐cells and T-­‐helper cells) appear not to cope with the abortive BPV-­‐infection in the tumor margins, possibly due to locally immune suppressive effects by CD4 + FOXP3 + RORgt – immunosuppressive Tregs. Furthemore, the role of BPV in ES, specifically definitive proof of infectivity and not just association, as well as the mechanisms of transformation, progression and transmission remain to be elucidated. The BPV-­‐genome consists of a double-­‐stranded, circular DNA, which persists episomally in infected cells. According to the currently favored theory, equine sarcoid disease is the only known cross-­‐species infection of papilloma viruses. However, infectious virions have never been isolated from a sarcoid tumor. Support for a suspected equid-­‐specific “(B)PV” infectious cycle has been provided by the epidemiology of ES in donkeys, which indicate a gradual spread in the absence of a bovine source. However, since both BPV-­‐1 and –2 are associated with ES, any specific changes (in sequence variation) would be expected to apply to both BPV-­‐1 and –2. Recently, a portion of the BPV genome sequence was identified that appears be “unique” for the equine isolates. Intriguingly, the identified deletion of these four residues within the L2 (late) protein will result in an identical motif in both BPV-­‐1 and -­‐2 variants. Whether or not this new motif is actually widespread within ES-­‐associated BPV-­‐isolates remains to be investigated. In collaboration with investigators from the Institute of Genetics and Virology in Berne and Zurich we are currently investigating the homology of papilloma virus DNA isolates from sarcoids and papillomas collected from affected horses, donkeys and cattle, respectively. The viral DNA was amplified with RCA and subsequently sequenced with Sanger sequencing. Funding: DKV Vetsuisse Bern; Agroscope; ISMEquine Research Main collaborations Vince Gerber, Eliane Marti, Gaudenz Dolf, Dominik Burger, Michael Stoffel, Tosso Leeb, Michaela Drögemüller, Giuseppe Bertoni (Bern) Derek Knottenbelt (Liverpool/Glasgow) Kurt Tobler, Sophie Ramsauer (Zürich) Techniques Immunofluorescence (IF), immunohistochemistry (IHC), quantitative PCR, Sanger Sequencing, rolling circle amplification (RCA) Peter KUHNERT, Institute of Veterinary Bacteriology peter.kuhnert@vetsuisse.unibe.ch Research interests Bacterial virulence, molecular epidemiology, phylogeny and taxonomy: thereby a broad variety of animal as well as zoonotic human pathogens are covered. It includes e.g. virulence mechanisms of Pasteurellaceae or genotyping of Mycoplasma hyopneumoniae, as well as source attribution and pathogenesis of Campylobacter, or pathotyping of Escherichia coli. Currently possible virulence mechanisms of the human gastroenteritis causing Campylobacter jejuni and Campylobacter coli are investigated. This includes genomic and proteomic comparison of clinical human isolates and commensal chicken strains. Furthermore their interaction with human cells is studied including invasion, cytotoxicity and cytokine induction. A second pillar of current research is dealing with transposon mutagenesis of the swine pathogen Mycoplasma hyopneumoniae. Using this approach factors involved in colonization and/or virulence should be identified and characterized. This will help to better understand the pathogenicity of this mycoplasma and could eventually lead to the generation of an attenuated vaccine. A continuous interest is in the characterization and taxonomy of Pasteurellaceae. This is often in the context of description of new species and genera from various animal species and helps in further understanding and diagnosis of animal bacterial diseases. Funding: Horizon 2020; BLV Main collaborations: Steve Djordjevic (Sydney) Henrik Christensen (Denmark) Dominik Maes (Ghent) Techniques General molecular biology including transposon mutagenesis, culture of mycoplasma, isolation and primary cultures of monocytes and lymphocytes, phylogenetic analyses, 2D Gel electrophoresis, real-­‐time PCR, Sanger sequencing, genotyping, molecular epidemiology, basic genome assembly Norbert MÜLLER, Institute of Parasitology norbert.mueller@vetsuisse.unibe.ch Research interests Chemotherapy of giardiasis: function of nitroreductases in the metabolism of nitro drugs metronidazole and nitazoxanide: the protozoan parasite Giardia lamblia causes the intestinal disease giardiasis that may lead to acute and chronic diarrhea in humans and various animal species. Nitro drugs such as the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are currently used for treatment of giardiasis. Our previous biochemical and molecular biological investigations showed that nitroreductases are important targets of both metronidazole and nitazoxanide. Furthermore, transgenic approaches in G. lamblia and Escherichia coli revealed that nitroreductases are important players in those mechanisms that are involved in the mode of action of, and resistance formation against, these nitro drugs. Our current experimentation is aimed at the elucidation of the biological function(s) of nitroreductases from G. lamblia. First assays demonstrated that these nitroreductases interact with key enzymes from the metabolism of G. lamblia. The nitroreductases are interactive and multifunctional most likely being not only involved in reduction of (toxic) nitro metabolites but also playing a major biological role in reduction of quinones (including ubiquinone) and FAD. Furthermore, our results suggested that the nitroreductases are closely associated to energy and intermediate metabolism of G. lamblia. By performing more detailed investigation of these interactions, we expect to learn not only more about the biology of the early eukaryote G. lamblia but gain also deeper insight into the mode of action of nitro drugs what, in turn, will favour the development of improved chemotherapeutic agents against G. lamblia and related parasites. Funding: Swiss National Science Foundation Main collaborations J. Müller, Institute of Parasitology, University of Bern A. Hemphill, Institute of Parasitology, Bern A. Hehl, Institute of Parasitology, Zürich C. Bovet/C. Largiadèr, Institute of Clinical Chemistry, Bern University Hospital, Berne. David Leitsch, Dept. of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria (currently on sabbatical in Bern) Selected publications •
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Nillius, D., Müller, J., Müller, N. (2011). Nitroreductase (GlNR1) increases susceptibility of Giardia lamblia and Escherichia coli to nitro drugs. Journal of Antimicrobial Chemotherapy, 66, 1029-­‐
1035. Müller J., Schildknecht P., Müller N. (2013) Metabolism of nitro drugs metronidazole and nitazoxanide in Giardia lamblia: characterization of a novel nitroreductase (GlNR2). Journal of Antimicrobial Chemotherapy 68, 1781-­‐1789 Müller J., Rout S., Leitsch D., Vaithilingam J.,, Hehl A., Müller N. (2015) Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds, International Journal for Parasitology: Drug and Drug Resistance 5, 37-­‐43 Vincent PERRETEN, Institute of Veterinary Bacteriology vincent.perreten@vetsuisse.unibe.ch Research interests Antimicrobial resistance and zoonosis: The emergence of multidrug-­‐resistant bacteria has become a challenge for both human and veterinary medicine representing one of the major public health hazards of the 21st century. In this regards close collaboration between both human and veterinary medicine has been established. A main task of the research group is to identify the antibiotic resistance genes and their location on genetic elements. However, some of them remain completely uncharacterized: the bacterial isolates show a specific antibiotic resistance phenotype, but the corresponding antibiotic resistance mechanism remains unknown. The last past years, several new antibiotic resistance genes have been discovered at the Institute of Veterinary Bacteriology: the new virginiamycin M1-­‐tiamulin-­‐clindamycin resistance gene [vga(E)] was identified in methicillin-­‐resistant Staphylococcus aureus (1), and three novel macrolide-­‐lincosamide-­‐streptogramin B resistance gene erm(43), erm(44) and erm(45) were identified in S. lentus, S. fleurettii, and S. xylosus, respectively (2,3,4). For instance, erm(44) was found to be located on a prophage, phage DNA which are integrated into the chromosome of bacteria (3). The group is also working on the identification of novel emerging pathogens (5). Additionally, larger epidemiological studies identify the molecular relationship between multi-­‐drug resistance plasmids spreading in Enterobateriaceae from animals and humans, as well as between methicillin-­‐resistant S. pseudintermedius and S. aureus from both animal and human origin. The research group has established technologies such as microarrays and next generation sequencing to characterize the different genetic elements and identify novel emerging pathogens and resistance mechanisms. Funding: SNF, ERA-­‐Net ANIHWA, BLV Main collaborations: Andrea Endimiani (University of Bern, Switzerland) Claude Favrot (University of Zurich, Switzerland) Stephen Kania, David Bemis (University of Tennessee, TN) Selected publications 1.
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Schwendener, S., and V. Perreten. 2011. New transposon Tn6133 in methicillin-­‐resistant Staphylococcus aureus ST398 contains vga(E), a novel streptogramin A, pleuromutilin, and lincosamide resistance gene. Antimicrob. Agents Chemother. 55(10):4900-­‐4904. Schwendener, S., and V. Perreten. 2012. New MLSB resistance gene erm(43) in Staphylococcus lentus. Antimicrob. Agents Chemother. 56(9):4746-­‐4752 Wipf, J.R., S. Schwendener, and V. Perreten. 2014. The novel MLSB resistance gene erm(44) is associated with a prophage in Staphylococcus xylosus. Antimicrob. Agents Chemother. 58(10):6133-­‐6138. Wipf, J.R., S. Schwendener, J.B. Nielsen, H. Westh, and V. Perreten. 2015. The new macrolide-­‐
lincosamide-­‐streptogramin B resistance gene erm(45) is located within a genomic island in Staphylococcus fleurettii. Antimicrob. Agents Chemother. 59(6):3578-­‐81. Gómez-­‐Sanz, E., S. Schwendener, A. Thomann, S. Gobeli Brawand, and V. Perreten. 2015. First SCCmec cassette containing mecB-­‐gene complex independent of transposon Tn6045 in a Macrococcus caseolyticus from a canine infection. Antimicrob. Agents Chemother. 59(8):4577-­‐
4583. Paola PILO, Institute of Veterinary Bacteriology paola.pilo@vetsuisse.unibe.ch Research interests Mechanisms of pathogenicity of mycoplasmas: Because of their minute genome, mycoplasmas are investigated as models for determining the minimal core genome necessary for self-­‐replicating cellular life and will play a central role in system biology determining microbe-­‐host interactions. Moreover, several mycoplasmal infections are emerging causing diseases in humans, animals and plants. We are currently using Mycoplasma bovis as a model organism to explore host-­‐
mycoplasmas interactions using high-­‐throughput technologies. We concentrate on two major aspects: a) cell permissivity and b) screening of genes involved in eukaryotic cell invasion. The project is divided in four parts: 1) development of in vitro infection models (investigated aspects are: cell tropism, endocytic pathways, cell invasion, cytotoxicity, cell proliferation and cell death (necrosis/apoptosis) 2) multiplex cytokines assay 3) construction of knockout mutant libraries of M. bovis, 4) high-­‐throughput screening of knockout mutant libraries for genes involved in cell invasion depending on host-­‐cell types using the gentamicin-­‐protection assay, deep sequencing and bioinformatics. Funding: Swiss National Science Foundation The biological cycle of zoonotic bacteria: It was recently estimated that 60% of all emerging infectious diseases are zoonoses. Most zoonoses are complex to investigate because of the involvement of several hosts and vectors linking the biological cycle of the causative agents to ecology and environmental factors. We are investigating the source, spread and persistence of zoonotic bacteria in a barn housing closely-­‐monitored free-­‐ranging house mice (Mus musculus domesticus) that experienced an outbreak of tularemia in 2012. The project aims to identify bacterial communities, highlighting zoonotic agents, in free-­‐ranging rodents, their ectoparasites and the surrounding environment using a metagenomic approach. Funding: Federal Office for the Environment Main collaborations Jose Perez-­‐Casal, Canada; Laurent Falquet, Fribourg; Michael H. Stoffel, Bern; Michèle Bodmer, Bern; Barbara König, Zürich Techniques Classical bacteriology, in vitro infection models, flow cytometry, fluorescence microscopy, NGS, molecular biology techniques, cell biology techniques. 5 recent publications •
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Bürki, S., Gaschen, V., Stoffel, M.H., Stojiljkovic, A., Frey, J., Kuehni-­‐Boghenbor, K., Pilo, P. 2015. Invasion and persistence of Mycoplasma bovis in embryonic calf turbinate cells. Vet. Res. 46:53. doi: 10.1186/s13567-­‐015-­‐0194-­‐z Bürki, S., Frey, J., Pilo, P. 2015. Virulence, persistence and dissemination of Mycoplasma bovis. Vet. Microbiol. 179(1-­‐2):15-­‐22. doi: 10.1016/j.vetmic.2015.02.024 Origgi, F.C., König, B., Lindholm, A.K., Mayor, D., Pilo, P. 2015. Tularemia among free-­‐ranging mice without infection of exposed humans, Switzerland, 2012. Emerg. Infect. Dis. 21(1):133-­‐5. doi: 10.3201/eid2101.140906 Origgi, F.C., Frey, J., Pilo, P. 2014. Characterisation of a new group of Francisella tularensis subsp. holarctica in Switzerland with altered antimicrobial susceptibilities, 1996 to 2013. Eurosurveillance. Jul 24;19(29). pii: 20858 •
Dean, A.S., Schelling, E., Bonfoh, B., Kulo, A.E., Boukaya, G.A., Pilo, P. 2014. Deletion in the gene BruAb2_0168 of Brucella abortus strains: diagnostic challenges. Clinical Microbiol. and Infection. 20(9): O550-­‐O553. doi: 10.1111/1469-­‐0691.12554 Sven ROTTENBERG, Institute of Animal Pathology sven.rottenberg@vetsuisse.unibe.ch Research interests DNA damage response and drug resistance in cancer: drug resistance is one of the most pressing hurdles in the clinic. Good examples are anti-­‐cancer drugs, some of which are also used to treat other diseases (e.g. protozoan and helminth infections). Despite the availability of various classical and novel anti-­‐cancer drugs, most patients with disseminated cancer are still dying because their tumors become resistant to all available drugs. In the context of anti-­‐cancer therapy, differences in the DNA damage response (DDR) provide a useful explanation for the initial drug sensitivity. To further explore defects in the DDR, exciting new opportunities for the development of targeted therapies have been achieved using the “synthetic lethal” approach: tumors that have lost critical DDR pathways rely more heavily on the remaining pathways, while normal host tissues still have all DDR pathways available. Inhibition of a remaining pathway in the DDR-­‐deficient cells will then cause lethality in tumor cells whereas the normal cells are not harmed. A successful example is the development of poly(ADP)ribose polymerase (PARP) inhibitors to kill BRCA1/2-­‐deficient cancer cells. In first clinical trials the preclinical observations also show a benefit of PARP inhibitors in patients that suffer from BRCA1/2-­‐deficient breast or ovarian cancers. As for many of the promising new therapies, however, resistance is an inevitable fact in clinical practice. Unfortunately, the precise mechanisms underlying resistance to classical chemotherapeutic drugs or to novel targeted drugs such as PARP inhibitors are poorly understood. In particular, little is known about BRCA1-­‐independent restoration of HR or how BRCA2-­‐deficient tumors adapt their DNA repair under treatment selection. To understand the basic mechanisms, we are putting emphasis on testing for “synthetic viability”: loss of function of another gene in addition to BRCA1 or BRCA2 will cause drug resistance. For this purpose we are performing loss-­‐of-­‐function screens in BRCA1/2 deficient tumor cells. In collaboration with the group of Jos Jonkers (NKI) we are inhibiting genes involved in the DDR using shRNA or gRNA (CRISPR/Cas9 technology) libraries. We have shown that this basic approach is useful to identify novel mechanisms of resistance. By understanding the drug resistance we also learn more about basic mechanisms of DNA repair, as we have just shown for the loss of REV7 and PARP inhibitor resistance. In collaboration with Thijn Brummelkamp (NKI) we are also performing drug resistance screens using haploid cells which have been mutagenized genome-­‐wide by the insertion of gene trap retroviruses. These screens have been highly successful to identify receptors involved in the entry of viruses or bacterial toxins into host cells. We are using them to identify mechanisms that may be important for drug uptake. For the validation of hits we are using genetically engineered mouse models for hereditary breast cancer in humans. Of these we have derived various cell lines and 3D cancer organoid cultures. Moreover, we are performing in silico analyses of data sets of human cancers and we are in close collaboration with clinical oncologists/pathologists to investigate samples from patients. Funding: Swiss National Science Foundation (SNSF); The Netherlands Organisation for Scientific Research (NWO) ; Dutch Cancer League (KWF) Main collaborations Jos Jonkers (The Netherlands Cancer Institute, NKI, Amsterdam): mouse models for human cancer, DDR-­‐targeting screens Thijn Brummelkamp (The Netherlands Cancer Institute, NKI, Amsterdam): Loss-­‐of-­‐
function screens using haploid cells Dan Durocher (Lunenfeld-­‐Tanenbaum Research Institute, Toronto) and Jiri Bartek (Danish Cancer Society Research Center, Copenhagen): DNA repair mechanisms Hans Clevers (Hubrecht Institute, The Netherlands): 3D cancer organoid cultures Aurel Perren (Institute of Pathology, Bern): Comparative Pathology 5 key publications •
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Xu et al. REV7 counteracts DNA double-­‐strand break resection and affects PARP inhibition. Nature 2015 [Epub ahead of print]. Jaspers et al. Loss of 53BP1 causes PARP inhibitor resistance in Brca1-­‐mutated mouse mammary tumors. Cancer Discovery 3:68-­‐81, 2013. Rottenberg et al. Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-­‐deficient mammary tumors. Cancer Research 72, 2350-­‐61, 2012 Pajic et al. Moderate increase in Mdr1a/1b expression causes in vivo resistance to doxorubicin in a mouse model for hereditary breast cancer. Cancer Research 69, 6396-­‐404, 2009. Rottenberg et al. High sensitivity of BRCA1-­‐deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proceedings of the National Academy of Science U S A (PNAS) 105, 17079-­‐17084, 2008. More information under http://www.nki.nl/divisions/molecular-­‐
pathology/rottenberg-­‐s-­‐group/ (new website at University of Bern is under development) Simone SCHULLER, Department clinical veterinary medicine simone.schuller@vetsuisse.unibe.ch Research interests Leptospirosis: epidemiology, diagnostics and pathogenic mechanisms: Leptospirosis is an important worldwide zoonosis affecting most mammalian species including dogs. Hosts become infected via contact of mucous membranes or broken skin with infected urine or contaminated water and soil. Infection with pathogenic leptospires can lead to a wide range of clinical manifestations, from a short self-­‐limiting febrile illness to life-­‐threatening disease often characterised by renal and hepatic compromise and haemorrhagic tendencies. In the past 20 years, severe pulmonary haemorrhage (Leptospiral Pulmonary Haemorrhage Syndrome, LPHS) has been reported with increasing incidence in humans and many other animal species including dogs, and has become a major cause of mortality. In our current research we are aiming to better understand the role of the dog in the epidemiology of leptospirosis in Switzerland, by evaluating the prevalence of anti-­‐leptospiral antibodies and urinary shedding of Leptospira spp in dogs not suspected to have leptospirosis. Secondly we are evaluating the diagnostic performance of patient side tests in the early diagnosis of leptospirosis in dogs. Thirdly we are investigating the role of direct interactions of pathogenic Leptospira with the host endothelia in the process of leptospiral organ dissemination and the breakdown of the alveolar barrier function seen in LPHS. Recent Publications 1-­‐4 1. Schuller S, Francey T, Hartmann K, et al. European consensus statement on leptospirosis in dogs and cats. J Small Anim Pract 2015;56:159-­‐179. 2. Schuller S, Callanan JJ, Worrall S, et al. Immunohistochemical detection of IgM and IgG in lung tissue of dogs with leptospiral pulmonary haemorrhage syndrome (LPHS). Comp Immunol Microbiol Infect Dis 2015;40:47-­‐53. 3. Schuller S, Sergeant K, Renaut J, et al. Comparative proteomic analysis of lung tissue from guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) reveals a decrease in abundance of host proteins involved in cytoskeletal and cellular organization. Journal of proteomics 2015;122:55-­‐72. 4. Juvet F, Schuller S, O'Neill EJ, et al. Urinary shedding of spirochaetes in a dog with acute leptospirosis despite treatment. Vet Rec 2011;168:564. Matthias SCHWEIZER, Institute of Virology and Immunology Federal Food Safety and Veterinary Office (FSVO) and University of Bern matthias.schweizer@vetsuisse.unibe.ch Research interests The interaction of pestiviruses with their host cells to manipulate and evade the innate and, therefore, also the adaptive immune system: For bovine viral diarrhea virus (BVDV), this might explain the success of this virus to cause persisting infection and immunotolerance and, thus, to persist in the host population. BVDV is a unique model to study many aspects of the virus in its natural setting. Due to the complete immune-­‐tolerance specific to the infecting virus strain, this model allows to thoroughly investigate the viral immune evasion mechanisms in the absence of adaptive immune responses. Additionally, we study a natural pathogen of cattle, and we are convinced that we can largely benefit from studying diseases caused by pathogens that have co-­‐
evolved with their hosts. One core area is the soluble viral glycoprotein Erns that exhibits an unusual RNase activity distinguished from the endogenous RNases present in bovine serum. Excitingly, these data may offer a new hypothesis for the role of Erns, i.e., it may prevent the host’s innate immune system from detecting the virus as “nonself” and inducing a strong interferon (IFN) response. Thus, pestiviruses might induce a form of “innate immunotolerance”, and this might also address more fundamental question that goes beyond that of the mechanism of a cattle disease, namely on the mechanisms that enable the host to differentiate between “self RNA” and “viral nonself RNA”. Accordingly, we have common projects to study persistent infections with small ruminant lentiviruses and to investigate the interaction of canine distemper virus (CDV, a paramyxovirus related the measles virus) with its host cells to evade innate immunity. As Swiss reference laboratory for pestiviruses, we participate in the development of diagnostics tests and in applied research projects to further understand transmission and pathogenesis of pestiviral infections. Funding: SNF; FSVO; IVI Main collaborations Till Rümenapf, Vienna (http://www.vetmeduni.ac.at/de/virologie/ ) Ueli Braun, Zurich (Farm Animals; http://www.tierspital.uzh.ch/Nutztiere.html) Claudia Bachofen, Zurich (Virology; http://www.vetvir.uzh.ch/) Philippe Plattet, Bern (Neurovirology; http://www.ekf.vetsuisse.unibe.ch/content/neurovirology/) Techniques Cell culture; standard virological, immunological and biochemical techniques; molecular biology; immunofluorescence; confocal microscopy; recombinant protein expression; quantitative (RT-­‐)PCR; flow cytometry. More information at http://www.ivv.unibe.ch/ and at http://www.ivi.ch Helmut SEGNER, Thomas WAHLI, Francesco ORIGGI, Centre for Fish and Wildlife Health helmut.segner@vetsuisse.unibe.ch Research interests Our research related to host-­‐pathogen interactions is focusing on four topics: 1. Interaction between the myxozoan parasite, Tetracapsuloides brysoalmonae, and salmonid fish: role of environmental temperature in modulating infection dynamics and the host immune response The myxozoan parasite, Tetracapsuloides brysoalmonae, causes the emerging Proliferative Kidney Disease (PKD) of salmonids like the rainbow trout, Oncorhynchus mykiss (main farmed species in Switzerland), and the brown trout, Salmo trutta fario (main wild species in Switzerland). At low water temperatures, under which the host-­‐parasite system has co-­‐evolved, trout are well able to survive the disease, while at increasing water temperatures, as they occur in the context of global warming, the disease leads to massive mortalities. Our research aims to unravel the processes underlying the temperature-­‐dependent increase of PKD-­‐induced mortalities. To test, we test two hypotheses: (i) levated water temperatures compromise the immune defense of the fish host against the parasite, and (ii) elevated water temperatures promote infectivity and virulence of the parasite towards the fish host. 2. Interaction between Aeromonas salmonicida and rainbow trout: Understanding immunosuppressive effects of the bacterium and immunoprotective responses of the fish as basis for the development of vaccines Aeromonas salmonicida is the etiological agent of furunculosis, an economically important disease of farmed salmonids. The bacterium possesses a complex type III secretion system which on the one hand is a key antigenic factor, and, on the other hand, appears to have immunosuppressive effects caused by the effector proteins that are secreted and translocated to the host cells. Our research, in collaboration ith the Institute of Veterinary Bacteriology, aims to characterize the role of the type III secretion system in the pathogen-­‐fish interaction, and to utilize this knowledge for vaccine development. 3. The immunomodulating role of endocrine factors on the immune system of rainbow trout and zebrafish Endocrine factors directly influence the immune system in order to fine-­‐tune immune investments within the physiological homeostasis and energy budget of the organism. It is well established that salmonids during the reproductive cycle are particularly susceptible to pathogen infections. In our research, we test the hypothesis that reproducing fish, as trade-­‐off between the energy costs of immunity and reproduction, downregulate their capacity to mount an immune repsonse to pathogen infections, and that this effect is mediated through treproductive sex steroids such as 17β-­‐estradiol. 4. The role of the plasticity of viral proteins for the virulence of the Canine Distemper Virus (CDV) and its ability to cross species barriers CDV is an important emerging pathogen in wildlife. The aim of this research is to understand how molecular interaction between viral ligand and host receptor proteins influence virulence and host specificity of CDV. To this end, in vitro studies are carried out with recombinant CDV attachment proteins and CDV chimeric genomes to examine the functionality and adaptability of viral proteins, including their role in host immune-­‐escape fitness of the virus. 5. Host pathogen interaction in the lower vertebrate battlefield Non-­‐fish lower vertebrates include reptiles and amphibians. These organisms are at the crossway of evolution and represent ideal animal models to investigate the evolution of host pathogen interaction. Complementing diagnostic pathology with molecular pathogenesis, immunology, phylogenetic analysis and next generation sequencing we investigate how genotypic variations redesign the pathology phenotype of disease in these animals. Main collaborations Joachim Frey, Bern: virulence/type III secretion system of Aeromonas salmonicida Hanna Hartikainen, Jukka Jukkela (Zürich): life cycle of Tetracapsuloides bryosalmonae Lidy van Kemenade (Wageningen), Magda Chadzinska (Krakau), Tiphaine Monsinjon (Le Havre): endocrine regulation of the fish immune system Phlippe Plattet, Bern: CDV virulence Elliott Jacobson (University of Florida): Host-­‐pathogen interaction in the lower vertebrate battlefield. Funding: SNF, SNF-­‐Sinergia, Euroepan Commission, Techniques Isolation and culture of fish, amphibian and reptilian cells Fish infection experiments and vaccine trials Histopathology, immunohistochemistry, in situ hybridization Routine serology-­‐based assays (serum neutralization, ELISA, western blots) Routine molecular techniques such as qPCR and cloning Flow cytometry Viral isolation and in vitro culture Site-­‐directed mutagenesis Recombinant protein expression and functional characterization More information under http://www.itpa.vetsuisse.unibe.ch/fiwi/ Artur SUMMERFIELD, Institute of Virology and Immunology artur.summerfield@vetsuisse.unibe.ch Research interests Understanding the cellular components of the innate immune system: a focus is placed on the mononuclear phagocytes system (MPS) of pigs, cattle and dogs, and its functioning during health and disease. The MPS is composed of a multitude of cell types and subsets, which can be characterized by their efficient ability to phagocytose, present and destroy antigen or pathogens. Despite this, the function of specific cell types is highly specialized, and at the same type characterized by functional plasticity. An example of our focus is phenotypical and functional characterization of classical and plasmacytoid dendritic cells and macrophages in the species mentioned above. Many projects in the group study the MPS in the context of the pathogenesis of virus infections. Furthermore, we aim to translate the knowledge acquired, in particular as it relates to dendritic cell biology, into development of improved vaccines or other therapeutics. Therein, we focus on TLR ligand selection and formulation to enhance innate immune responses, ultimately resulting in improved adaptive responses, both systemically and at mucosal surfaces. The viral models currently investigated include classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-­‐and-­‐mouth disease virus (FMDV), Japanese encephalitis virus and bovine respiratory syncytial virus (BRSV). Current funding: EU FP7, Horizon 2020, SNF, ERA-­‐Net ANIHWA, industrial partners Main collaborations Nicolas Ruggli et al., Mittelhäusern: CSFV and PRRSV pathogenesis and vaccines, Remy Bruggmann et al, Bern: cell transcriptomics using RNA sequencing Predemics Consortium (FP7 project) Saphir Consortium (Horizon 2020 project) KILLeuPRRSV Consortium (Anihwa) Techniques All techniques used in cellular immunology including cell sorting and flow cytometry for cell isolation, phenotyping, functional tests, proliferation, cytokine release. Cell culture, T-­‐cell assays, ELISA. Monoclonal antibody technology. Transcriptomic profiling using RNA sequencing. Virological techniques. Selected publications Summerfield A., Auray G., Ricklin M.E. (2015). Comparative dendritic cells biology of veterinary mammals.
Annual Review Animal Biosciences. Volume 3.
Baumann A., Démoulins T., Python S., Summerfield A. 2014. Porcine cathelicidins efficiently complex and
deliver nucleic acids to plasmacytoid dendritic cells and can thereby mediate bacteria-induced IFN-α
responses. J. Immunol 193:364.
Baumann A, Mateu, E, Murtaugh MP, Summerfield A (2013). Impact of genotype 1 and 2 of porcine
reproductive and respiratory syndrome viruses on interferon-alpha responses by plasmacytoid dendritic
cells. Vet Res 44 (1), 33.
Python S., Gerber M. Sutter R., Ruggli, N. and Summerfield A. 2013. Efficient sensing of infected cells in
absence of virus particles by plasmacytoid dendritic is blocked by the viral ribonuclease Erns.
PlosPathogens 9(6):e1003412
Saurer, L., McCullough, K.C., Summerfield, A. In vitro induction of mucosa-type dendritic cells by all-trans
retinoic acid. J. Immunol. 179:3504.
Meurens F, Summerfield A, Nauwynck H, Saif L, Gerdts V. (2012). The pig: a model for human infectious
diseases. Trends in microbiology 20 (1), 50-57
Volker THIEL, Institute for Virology and Immunology (IVI) volker.thiel@vetsuisse.unibe.ch Research interests Coronavirus-­‐host interaction; virus innate immune evasion; coronavirus replication. Over the past years the research group of Dr. Thiel has been able to clone and propagate full length coronavirus (CoV) cDNAs from several CoVs using vaccinia virus as eukaryotic cloning vector. The reverse genetic system is based upon the in vitro transcription of infections RNA from a cloned full-­‐length cDNA copy of a CoV genome, and the introduction of nucleotide changes, deletions or insertions is facilitated by vaccinia virus-­‐mediated recombination. The reverse genetic system has proven to be rapid, robust and versatile and is available in the laboratory of Dr. Thiel for the generation of recombinant prototype viruses of all major CoV phylogenetic lineages, namely for HCoV 229E, type-­‐I and type-­‐II Feline CoVs (genus Alphacoronavirus), Mouse Hepatitis Virus strain A59, SARS-­‐CoV (genus Betacoronavirus), Avian Infectious Bronchitis Virus (genus Gammacoronavirus). One of our long-­‐term goals is to study CoV replication in order to develop strategies to prevent and control CoV infections. Thus, we have applied the reverse genetic system to the analysis of mechanisms and enzymes involved in CoV genome expression on the molecular level. We could identify and characterize mechanisms of translational regulation of CoV gene expression, such as internal ribosomal entry on subgenomic mRNAs, and ribosomal frame-­‐shifting on the CoV genomic RNA. Furthermore, we have studied enzymatic activities encoded by the CoV replicase gene and analyzed CoV replication, transcription and packaging using recombinant CoVs, replicon RNAs, defective minigenomes, and virus-­‐like particles. We have extended these studies to the identification and evaluation of CoV replicase inhibitors, the analysis of cis-­‐acting elements involved in RNA replication, the analysis of CoV target cell tropism, the generation of CoV-­‐based vaccine vectors, the analysis of virus-­‐host interactions in vivo using a murine model of CoV infection, and in depths analyses of virus-­‐induced innate immune responses. In order to study virus-­‐host interactions of respiratory viruses we have recently established primary airway epithelial cultures that grow under so-­‐called “air-­‐liquid-­‐interface” conditions. This culture system is now available for several species (human, camelid, bats) and allows us to characterize particular virus-­‐host interactions (e.g. innate immune evasion), and zoonotic transmission of CoVs in an environment that resembles the authentic primary target tissue of CoV infection. Funding: SNF; DFG; BLV Main collaborations Stefan Kunz (CHUV, Lausanne) & Stephen Leib (IFIK; Bern): Tropism, pathogenesis, and innate immune evasion of emerging viruses Christian Drosten and Felix Drexler (Virology, Bonn, Germany): Evolutionary aspects of coronavirus zoonotic transmission Rune Hartmann (Structural Biology, Aarhus, Denmark): Interferon lambda Ronald Dijkman (IVI, Bern): Primary airway epithelial cultures Edward Trybala (Infectious Medicine, Göteborg, Sweden): Coronavirus inhibitors Nicolas Ruggli (IVI, Mittelhäusern): anti-­‐PRRSV drugs Techniques Basic and advanced virological techniques; reverse genetics; cell cutlure; airway epithelial cultures; lentiviral vectors; EM and confocal microscopy; basic molecular biological techniques Selected publications •
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Lundin A., Dijkman R., Bergström T., Kann N., Adamiak B., Hannoun C., Kindler E., Jónsdóttir H.R., Muth D., Kint J., Forlenza M., Müller M.A., Drosten C., *Thiel V. , and E. Trybala* (2014). Targeting membrane-­‐bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus. PLoS Pathog. 10(5):e1004166. *shared senior authorship Kindler E., and V. Thiel (2014). To sense or not to sense viral RNA-­‐-­‐essentials of coronavirus innate immune evasion. Curr Opin Microbiol. 20:69-­‐75. Hamming O.J., Terczyń ska-­‐Dyla E., Vieyres G., Dijkman R., Jørgensen S.E., Akhtar H., Siupka P., Pietschmann T., Thiel V. , and R. Hartmann (2013). Interferon lambda 4 signals via the IFNλ receptor to regulate antiviral activity against HCV and coronaviruses. EMBO J. 32(23):3055-­‐65. Raj, V.S., Mou, H., Smits, S.L., Dekkers, D.H., Müller, M.A., Dijkman, R., Muth, D., Demmers, J.A., Zaki, A., Fouchier, R.A., Thiel, V. , Drosten, C., Rottier, P.J., Osterhaus, A.D., Bosch, B.J., and B.L.Haagmans (2013). Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-­‐EMC. Nature 495(7440):251-­‐4. Kindler, E., Jónsdóttir, H.R., Muth, D., Hamming, O.J., Hartmann, R., Rodriguez, R., Geffers, R., Fouchier, R.A., Drosten, C., Müller, M.A., Dijkman, R., and V. Thiel (2013). Efficient Replication of the Novel Human Betacoronavirus EMC on Primary Human Epithelium Highlights Its Zoonotic Potential. MBio. 4(1). doi:pii: e00611-­‐12. Zust, R., Cervantes-­‐Barragan, L., Habjan, M., Maier, R., Neuman, B. W., Ziebuhr, J., Szretter, K. J., Baker, S. C., Barchet, W., Diamond, M. S., Siddell, S. G., Ludewig, B. & Thiel, V. (2011). Ribose 2'-­‐O-­‐
methylation provides a molecular signature for the distinction of self and non-­‐self mRNA dependent on the RNA sensor Mda5. Nature Immunology 12, 137-­‐43. Daffis, S., Szretter, K. J., Schriewer, J., Li, J., Youn, S., Errett, J., Lin, T. Y., Schneller, S., Zust, R., Dong, H., Thiel, V. , Sen, G. C., Fensterl, V., Klimstra, W. B., Pierson, T. C., Buller, R. M., Gale, M., Jr., Shi, P. Y. & Diamond, M. S. (2010). 2'-­‐O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468 , 452-­‐6. Cervantes-­‐Barragan, L., Zust, R., Maier, R., Sierro, S., Janda, J., Levy, F., Speiser, D., Romero, P., Rohrlich, P. S., *Ludewig, B. & *Thiel, V. (2010). Dendritic cell-­‐specific antigen delivery by coronavirus vaccine vectors induces long-­‐lasting protective antiviral and antitumor immunity. MBio 1(4). pii: e00171-­‐10. *shared senior authorship. Bocharov, G., Zust, R., Cervantes-­‐Barragan, L., Luzyanina, T., Chiglintsev, E., Chereshnev, V. A., *Thiel, V. & *Ludewig, B. (2010). A systems immunology approach to plasmacytoid dendritic cell function in cytopathic virus infections. PLoS Pathog 6, e1001017. *shared senior authorship. Züst, R., Cervantes-­‐Barragán, L., Kuri, T., Blakqori, G., Weber, F., Ludewig, B., & Thiel V. (2007) Coronavirus non-­‐structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines. PLoS Pathog. 3(8):e109. More information: http://www.blv.admin.ch/ivi/index.html; http://www.ivv.unibe.ch Kerry WOODS, Institute of Animal Pathology kerry.woods@vetsuisse.unibe.ch Research interests Molecular interactions between the Theileria schizont and its host cell: the Woods research group is a new group within the ITPA as of 1 November 2014. We are interested in the molecular interactions that occur between the intracellular parasite Theileria, and its bovine host cell. Theileria annulata is the causative agent of Tropical Theileriosis, an important disease of cattle that is prevalent throughout the Mediterranean basin, North Africa and parts of Asia. In addition to being a pathogen of economic importance, the cell biology underlying Theileria infection is quite fascinating. The Theileria schizont “transforms” the bovine leukocytes that it infects, immortalizing the cell and conferring upon it a leukaemia-­‐like phenotype. T. annulata transformed macrophages proliferate continually and can be maintained in culture indefinitely. Theileria-­‐induced transformation can be reversed upon killing the parasite, making these infected cells a versatile and reversible system with which to study cell signaling pathways associated with cancer. The strictly intracellular parasite resides in the host cell cytoplasm, and ensures its persistence within these immortalized cells by interacting with host cell microtubules and “incorporating” itself within the central spindle during host cell mitosis and cytokinesis. In this way, the parasite is distributed to both daughter cells during host cell division. Our main interest is in the study of host-­‐parasite interactions that occur at the schizont surface, with the hypothesis that the parasite surface acts as a signal transduction platform that contributes to Theileria-­‐induced transformation. In particular, we are focusing on host cell microtubule-­‐associated proteins and adaptor proteins that we found to coat the parasite surface. We use live cell imaging and immuno-­‐fluorescence to analyse the dynamics of host-­‐parasite interactions, and a range of techniques (including co-­‐immunoprecipitation and proximity-­‐dependent biotin identification) to detect protein-­‐protein interaction networks. The functional significance of such host-­‐parasite interactions will be investigated by targeting host proteins using shRNA or CRISPR/Cas9 technology. Funding: Swiss National Science Foundation (Ambizione) Selected Publications •
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Wiens, O., Xia, D., von Schubert, C., Wastling, JM., Dobbelaere, DAE., Heussler, VT., Woods, KL. Cell Cycle-­‐dependent Phosphorylation of Theileria annulata Schizont Surface Proteins. Plos One. 2014;9(7):e103821. Woods, KL., Theiler, R., Mühlemann, M, Segiser, A, Huber, S., Ansari, H.R, Pain, A., Dobbelaere, D. 2013. Recruitment of EB1, a master regulator of microtubules, by the Theileria annulata schizont. Plos Pathogens. Woods, KL., von Schubert, C., Dobbelaere, D. 2013. Protein Phosphorylation in Parasites. Chapter 9. Willey Publishers. Von Schubert, C., Xue, G., Schmuckli-­‐Maurer, J., Woods, KL., Nigg, EA., Dobbelaere, (2010). The Transforming Parasite Theileria Co-­‐opts Host Cell Mitotic and Central Spindles to Persist in Continually dividing cells. Plos Biology.