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The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Immunopathogenesis of postnatal persistent infection generated by classical swine fever virus and its implication in the superinfection exclusion phenomenon Sara Muñoz González PhD Thesis Bellaterra, 2017 Immunopathogenesis of postnatal persistent infection generated by classical swine fever virus and its implication in the superinfection exclusion phenomenon Tesis doctoral presentada por Sara Muñoz González para acceder al grado de doctor en el marco del programa de Doctorado en Medicina y Sanidad Animal de la Facultat de Veterinaria de la Universitat Autònoma de Barcelona, bajo la dirección de la Dra. Llilianne Ganges Espinosa y del Dr. Mariano Domingo Álvarez. Bellaterra, 2017 This work has been financially supported by grant AGL2012-38343 from Spanish government Sara Muñoz-González received predoctoral fellowship FI-DGR 2014 from AGAUR, Generalitat de Catalunya. La Dra Llilianne Ganges Espinosa, investigadora del Centre de Recerca de Sanidad Animal y del Instituto de Investigación Agroalimentaria (CReSA-IRTA), y el y el Dr. Mariano Domingo Álvarez, Catedrático de Universidad del Área de Conocimiento de Sanidad Animal perteneciente al Departamento de Sanitat i Anatomia Animals en la Universitat Autònoma de Barcelona (UAB), adscrito como investigador al CReSA-IRTA Certifican: Que la memoria titulada « Immunopathogenesis of postnatal persistent infection generated by classical swine fever virus and its implication in the superinfection exclusion phenomenon » presentada por Sara Muñoz González para la obtención del grado de Doctora en Medicina y Sanidad Animales, se ha realizado bajo su supervisión y tutoría, y autorizan su presentación para que sea valorada por la comisión establecida. Para que conste a los efectos oportunos, firman el presente certificado en Bellaterra (Barcelona), el 17 de febrero de 2017. Dra. Llilianne Ganges Espinosa Dr. Mariano Domingo Álvarez Directora Director y tutor Sara Muñoz González Doctoranda TABLE OF CONTENTS Table of contents i List of abbreviations iii Abstract vii Resumen ix PART I - General introduction and objectives .......................................... 1 Chapter 1 - General introduction ....................................................................... 3 1.1 Classical swine fever: distribution and economic impact along the history ................................................................................................................ 5 1.2 Classical swine fever virus ..................................................................... 7 1.2.1 Physicochemical properties and taxonomy .................................... 7 1.2.2. Genome organization and expression ............................................. 8 1.3 Pathogenesis ......................................................................................... 13 1.3.1. Virus-host interaction ................................................................... 14 1.3.2. Humoral and cellular responses in CSFV infection ..................... 19 1.4 Clinical forms of CSF .......................................................................... 21 1.4.1. CSF acute form ............................................................................. 22 1.4.2. CSF chronic form ......................................................................... 23 1.4.3. CSF congenital persistent form .................................................... 24 1.5 Epidemiology ....................................................................................... 26 1.5.1. Transmission and spread .............................................................. 26 1.5.2 Molecular epidemiology .............................................................. 29 1.5.3. Control and surveillance ............................................................... 31 1.6 Diagnosis and related diagnostic tools ................................................. 32 1.7 Vaccine strategies against CSFV ......................................................... 36 Chapter 2 - Objectives ....................................................................................... 41 i PART II - Studies ........................................................................................... 45 Chapter 3 - Study I: Postnatal persistent infection with classical swine fever virus and its immunological implications............................................................ 47 Chapter 4 - Study II: Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs ................................................ 71 Chapter 5 - Study III: Classical swine fever virus vs. classical swine fever virus: The superinfection exclusion phenomenon in experimentally infected wild boar. ............................................................................................................. 83 PART III - General discussion and conclusions ...................................... 96 Chapter 6 - General discussion ...................................................................... 107 Chapter 7 - Conclusions .................................................................................. 121 References ....................................................................................................... 125 ii LIST OF ABBREVIATIONS APPV Atypical porcine pestivirus ASF African swine fever BDV Border disease virus BMHC Bone marrow hematopoietic cells BSL3 Biosecurity level 3 BVDV Bovine viral diarrhea virus CD Cluster differentiation CNS Central nervous system Cp Cytopathogenic CSF Classical swine fever CSFV Classical swine fever virus CTL Cytotoxic T lymphocyte DAS Double-antibody-sandwich DC Dendritic cell DIC Disseminated intravascular coagulation DIVA Differentiation of infected from vaccinated animals DPI Days post infection DPV Days post vaccination EDC Endothelial cells ELISA Enzyme-linked immunosorbent assays EU European Union EURL European Union Reference Laboratory iii FAT Fluorescent antibody test HC Hog cholera (now classical swine fever) IFN Interferon IL Interleukin IRES Internal Ribosome Entry Site ISG IFN stimulated genes MΦ Macrophage NPLA Neutralizing peroxidase–linked assay NrPV Norwegian rat pestivirus OIE World Organisation for Animal Health ORF Open reading frame PBMC Peripheral blood mononuclear cell pDC Plasmocytoid dendritic cell PDNS Porcine Dermatitis and Nephropathy Syndrome PHA Phytohaemagglutinin PI Persistently infected PLA Peroxidase-linked assay RaPestV Rhinolophus affinis bats RNA Ribonucleic acid RT-PCR Reverse transcription-polymerase chain reaction SIE Superinfection exclusion SLA Swine Leucocyte Antigens or histocompatibility complex (MHC) proteins TSV Tunisian sheep virus iv UTR Untranslated regions v vi ABSTRACT Classical swine fever (CSF) is a highly contagious viral disease of domestic and wild pigs, included in the list of diseases notifiable to the World Organisation for Animal Health. The causative agent, CSF virus (CSFV), belongs to the Pestivirus genus, Flaviviridae family. Over the last century, great efforts have been directed towards the control and eradication of CSF, which today remains one of the most important diseases for animal health and in the pig industry worldwide. The circulation and intrinsic importance of low and moderate virulence strains in the endemic regions has been extensively described. Also, it has been shown the role of these type of viral strains in the generation of “the pregnant carrier sow syndrome” and persistent infection of piglets after trans-placental transmission. However, the mechanisms involved in this form of CSFV infections are not well known, and the existing studies date over 40 years ago. By contrast, the possible generation of viral persistence after postnatal infection of pigs is still a question to resolve. Against this background, this thesis has demonstrated the capacity of low and moderate virulence CSFV strains to produce postnatal persistent infection early after birth. To this end, newborn piglets were inoculated intranasally with CSFV. During six weeks, these piglets remained apparently healthy, although they were not able to generate detectable CSFV specific humoral nor cellular immune responses, maintaining high virus load in blood, organs and body secretions and excretions. In addition, it has been demonstrated the ineffectiveness of vaccination of six-week-old PI pigs, which were unable to elicit a detectable innate immune response, in terms of IFN type-I production, as well as acquired immune responses (i.e. IFN type-II and antibodies) following vaccination with a CSFV live attenuated vaccine (C-strain). The RNA of the vaccine could not be detected by a specific RT-qPCR in any of the samples analysed after vaccination, including the tonsil, suggesting a superinfection exclusion (SIE) phenomenon between the persistently infecting virus (primary infection) and the CSFV vii vaccine strain (secondary infection). Finally,