Institute of Virology and Immunoprophylaxis


Institute of Virology and Immunoprophylaxis


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Guzylack-Piriou L.,French National Institute for Agricultural Research | Alves M.P.,Institute of Virology and Immunoprophylaxis | McCullough K.C.,Institute of Virology and Immunoprophylaxis | Summerfield A.,Institute of Virology and Immunoprophylaxis
Developmental and Comparative Immunology | Year: 2010

Based on the known importance of Flt3 ligand (Flt3L) for the development of mouse dendritic cells (DCs), the present study compared the phenotype and function of DC derived from porcine bone marrow haematopoietic cells using either granulocyte-macrophage colony-stimulating factor or Flt3L (GMCSF-DC and Flt3L-DC, respectively). To this end, porcine Flt3L was cloned resulting in the identification of three isoforms of Flt3L. Compared to GMCSF-DC which were uniformly CD14+, Flt3L-DC had a more diverse phenotype comprised of CD172a-CD11a- progenitor cells, CD172a+CD14-CD163- DC and CD172a+CD14+CD163+ DC. In addition, only the Flt3L-DC contained interferon-producing plasmacytoid DC, although their frequency was low. Only the CD14- Flt3L-DC responded to TLR2, -3, -4, -7 and -9 agonists by upregulating CD80/86. This population of DC was also more potent in T-cell stimulation assays when compared to the CD14+ counterpart. Interestingly, Flt3 was not only highly expressed on DC precursors, but also found on Flt3L-DC but not on GMCSF-DC or monocyte-derived DC. Furthermore, also DC circulating in the blood but not monocytes or other leukocytes expressed this receptor. Taken together, our study demonstrates that Flt3L-DCs are more suitable to study the interaction of pathogens with DC. Moreover, we show that also in the pig Flt3 remains expressed in a restricted manner on DC originating from a bone marrow DC precursors, typically representing steady-state DC in lymphoid tissue and blood. © 2009 Elsevier Ltd. All rights reserved.

Saiz J.-C.,Instituto Nacional Of Investigacion Y Tecnologia Agraria Y Alimentaria Inia | McCullough K.C.,Institute of Virology and Immunoprophylaxis
Virus Research | Year: 2012

Virus infection of host cells requires that entry into the cell results in efficient genome release leading to translation and replication. These initial steps revolving around the entry and genomic release processes are crucial for viral progeny generation. Despite the variety of receptors used by viruses to initiate entry, evidence from both enveloped and non-enveloped viral infections is highlighting the important role played by intracellular acidic compartments in the entry of many viruses. These compartments provide connecting nodes within the endocytic network, presenting multiple viral internalization pathways. Endosomal compartments employing an internal acidic pH can trigger molecular mechanisms leading to disassembly of viral particles, thus providing appropriate genome delivery. Accordingly, viruses have evolved to select optimal intracellular conditions for promoting efficient genome release, leading to propagation of the infectious agent. This review will address the implications of cellular compartment involvement in virus infectious processes, and the roles played by the viruses' own machinery, including pH sensing mechanisms and the methodologies applied for studying acid-dependent viral entry into host cells. © 2012 Elsevier B.V.

Tappeiner C.,Inselspital | Flueckiger F.,Institute of Virology and Immunoprophylaxis | Boehnke M.,Institute of Ophthalmology | Goldblum D.,University of Basel | Garweg J.G.,University of Bern
Journal of Cataract and Refractive Surgery | Year: 2012

Purpose: To assess the impact of topical anesthetic agents and ethanol on ocular surface wound healing using an ex vivo whole-globe porcine model. Setting: Department of Ophthalmology, Inselspital, University of Bern, Bern, Switzerland. Design: Experimental study. Methods: Standardized corneoepithelial lesions (5.0 mm diameter, 40 μm depth) were created with excimer laser light in freshly enucleated porcine eyes. The globes (6 per group) were exposed to different concentrations of ethanol (2.0% to 99.0%), cocaine (2.0% to 10.0%), procaine hydrochloride (0.4%), tetracaine (0.5% to 1.0%), or lidocaine (2.0%), 3 drops/hour for 3 hours. Control solutions were physiologic saline, balanced salt solution, and tissue-culture medium. After 20 to 26 hours, wound-healing response was compared by measuring the diameter of each corneoepithelial lesion. Results: The mean diameter of corneoepithelial lesions exposed to physiologic saline decreased from 4.78 mm ± 0.19 (SD) to 4.44 ± 0.17 mm between 20 and 26 hours. After 24 hours, the mean lesion size, compared with physiological saline, was larger after cocaine 5.0% (5.20 ± 0.26 mm) and 10.0% (5.39 ± 0.12 mm), tetracaine 0.5% (5.59 ± 0.35 mm) and 1.0% (5.55 ± 0.27 mm), and procaine hydrochloride 0.4% (5.76 ± 0.12 mm), but not after lidocaine 2.0% (5.01 ± 0.17 mm). Balanced salt solution, tissue-culture medium, ethanol 2.0% to 99.0%, and cocaine 2.0% did not inhibit the wound-healing response. Conclusions: In an ex vivo whole-globe porcine model, lidocaine 2.0% and cocaine 2.0% were the least toxic anesthetic agents. At all concentrations, ethanol had no impact on wound healing. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. © 2012 ASCRS and ESCRS.

Switzerland had been affected by the bluetongue virus serotype 8 (BTV-8) epidemic in Europe in the years 2007 to 2009. After three years of mandatory vaccination and comprehensive surveillance, Switzerland showed to be free of BTV-8 in 2012. In the future Elisa testing of bulk-tank milk (BTM) samples as a very sensitive and cost-effective method should be used for the surveillance of all serotypes of BTV. To determine the prevalence of seropositive herds, BTM from 240 cattle herds was sampled in July 2012. The results showed an apparent seroprevalence of 98.7% in the investigated dairy herds. Most plausible, the high prevalence was caused by the vaccination campaigns rather than by infections with BTV-8. In the outbreak the cumulative number of BTV-8 cases in Switzerland had been 75. Thus it is very likely that the used inactivated vaccines induced long-term antibody titres. Due to the high seroprevalence, investigating for BT-antibodies cannot be used for early recognition of a new introduction of BTV at the moment. Nonetheless, testing of BTM samples is appropriate for an annual evaluation of the seroprevalence and especially as an instrument for early recognition for incursions as soon as the antibody prevalence declines. To determine this decline the BTM testing scheme should be conducted each year as described in this work. © 2014 Schlütersche Verlagsgesellschaft mbH & Co. KG.

Haines F.J.,Animal Health and Veterinary Laboratories Agency | Hofmann M.A.,Institute of Virology and Immunoprophylaxis | King D.P.,The Pirbright Institute | Drew T.W.,Animal Health and Veterinary Laboratories Agency | Crooke H.R.,Animal Health and Veterinary Laboratories Agency
PLoS ONE | Year: 2013

A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping. © 2013 Haines et al.

Ricklin M.E.,University of Bern | Roosje P.,University of Bern | Summerfield A.,Institute of Virology and Immunoprophylaxis
Journal of Clinical Immunology | Year: 2010

Atopic dermatitis in humans and dogs is a chronic relapsing allergic skin disease. Dogs show a spontaneous disease similar to the human counterpart and represent a model to improve our understanding of the immunological mechanisms, the pathogenesis of the disease, and new therapy development. The aim of the study was to determine the frequency and phenotype of dendritic cells (DC) in the epidermis and dermis of healthy, canine atopic dermatitis lesional, and non-allergic inflammatory skin to further validate the model and to obtain insights into the contribution of DC to the pathogenesis of skin diseases in dogs. We first characterized canine skin DC using flow-cytometric analysis of isolated skin DC combined with an immunohistochemical approach. A major population of canine skin dendritic cells was identified as CD1c +CD11c+CD14-CD80+MHCII+- MAC387- cells, with dermal DC but not Langerhans cells expressing CD11b. In the epidermis of lesional canine atopic dermatitis and non-allergic inflammatory skin, we found significantly more dendritic cells compared with nonlesional and control skin. Only in canine atopic dermatitis skin did we find a subset of dendritic cells positive for IgE, in the epidermis and the dermis. Under all inflammatory conditions, dermal dendritic cells expressed more CD14 and CD206. MAC387+ putative macrophages were absent in healthy but present in inflamed skin, in particular during non-allergic diseases. This study permits a phenotypic identification and differentiation of canine skin dendritic cells and has identified markers and changes in dendritic cells and macrophage populations related to allergic and nonallergic inflammatory conditions. Our data suggest the participation of dendritic cells in the pathogenesis of canine atopic dermatitis similar to human atopic dermatitis and further validate the only non-murine spontaneous animal model for this disease. © Springer Science+Business Media, LLC 2010.

Sharma R.,Institute of Virology and Immunoprophylaxis | Ghasparian A.,University of Zürich | Robinson J.A.,University of Zürich | McCullough K.C.,Institute of Virology and Immunoprophylaxis
PLoS ONE | Year: 2012

DC employ several endocytic routes for processing antigens, driving forward adaptive immunity. Recent advances in synthetic biology have created small (20-30 nm) virus-like particles based on lipopeptides containing a virus-derived coiled coil sequence coupled to synthetic B- and T-cell epitope mimetics. These self-assembling SVLP efficiently induce adaptive immunity without requirement for adjuvant. We hypothesized that the characteristics of DC interaction with SVLP would elaborate on the roles of cell membrane and intracellular compartments in the handling of a virus-like entity known for its efficacy as a vaccine. DC rapidly bind SVLP within min, co-localised with CTB and CD9, but not caveolin-1. In contrast, internalisation is a relatively slow process, delivering SVLP into the cell periphery where they are maintained for a number of hrs in association with microtubules. Although there is early association with clathrin, this is no longer seen after 10 min. Association with EEA-1+ early endosomes is also early, but proteolytic processing appears slow, the SVLP-vesicles remaining peripheral. Association with transferrin occurs rarely, and only in the periphery, possibly signifying translocation of some SVLP for delivery to B-lymphocytes. Most SVLP co-localise with high molecular weight dextran. Uptake of both is impaired with mature DC, but there remains a residual uptake of SVLP. These results imply that DC use multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raft-mediated macropinocytosis. With most SVLP-containing vesicles being retained in the periphery, not always interacting with early endosomes, this relates to slow proteolytic degradation and antigen retention by DC. The present characterization allows for a definition of how DC handle virus-like particles showing efficacious immunogenicity, elements valuable for novel vaccine design in the future. © 2012 Sharma et al.

Bruckner L.,Institute of Virology and Immunoprophylaxis
Biologicals | Year: 2010

Freedom from extraneous agents is a high priority for any medicinal product. For veterinary vaccines, extraneous agents testing is addressed by different regulations of the European Pharmacopoeia and guidelines issued by the European Medicines Evaluation Agency. This article provides a brief review of the relevant texts. © 2010 The International Association for Biologicals.

Lannes N.,Institute of Virology and Immunoprophylaxis | Summerfield A.,Institute of Virology and Immunoprophylaxis
PLoS ONE | Year: 2013

Plasmacytoid dendritic cells (pDC) are the most potent producers of type-I interferon (IFN) and represent the main interferon (IFN)-α source in response to many viruses. Considering the important roles played by type I IFN's, not only as antiviral effectors but also as potent alarming cytokine of the immune system, we investigated how such responses are regulated by various cytokines. To this end, we stimulated enriched pDC in the presence or absence of particular cytokines with a strong activator, CpG DNA, or a weak activator of pDC, foot-and-mouth disease virus (FMDV). Alternatively, we pre-incubated pDC for 16 h before stimulation. The pro-inflammatory cytokines tested Interleukin (IL)-6, IL17A, tumour necrosis factor (TNF)-α did not influence IFN-α responses except TNF-α, which promoted responses induced by FMDV. The haematopoietic cytokines Fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) had enhancing effects on pDC activation at least in one of the protocols tested. IFN-β and IFN-γ were the most potent at enhancing FMDV-induced IFN-α, up to 10-fold. Interestingly, also the Th2 cytokine IL-4 was an efficient promoter of pDC activity, while IL-10 was the only negative regulator of IFN-α in pDC identified. The cytokines enhancing IFN-α responses also promoted pDC survival in cell culture with the exception of GM-CSF. Taken together this work illustrates how the cytokine network can influence pDC activation, a knowledge of relevance for improving vaccines and therapeutic interventions during virus infections, cancers and autoimmune diseases in which pDC play a role. © 2013 Lannes, Summerfield.

Bruckner L.,Institute of Virology and Immunoprophylaxis
Journal of Comparative Pathology | Year: 2010

In Europe, two main regulatory texts regulate the immunogenicity requirements of vaccines, the European Pharmacopoeia and the guidelines of the European Medicines Evaluation Agency (EMEA). This article outlines these requirements and the regulatory challenges. © 2009 Elsevier Ltd. All rights reserved.

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