Unravelling the Pathobiological Diversity of Highly Pathogenic Avian Influenza in Birds
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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 UNRAVELLING THE PATHOBIOLOGICAL DIVERSITY OF HIGHLY PATHOGENIC AVIAN INFLUENZA IN BIRDS Raúl Sánchez González PhD Thesis Bellaterra, 2019 Unravelling the pathobiological diversity of highly pathogenic avian influenza in birds Tesi doctoral presentada per Raúl Sánchez González per optar al grau de Doctor en Veterinària dins del programa de doctorat de Medicina i Sanitat Animals del Departament de Sanitat i d’Anatomia Animals de la Facultat de Veterinària de la Universitat Autònoma de Barcelona, sota la direcció de la Dra. Natàlia Majó i Masferrer i el Dr. Antoni Ramis Salvà. Bellaterra, 2019 La Dra. NATÀLIA MAJÓ i MASFERRER i el Dr. ANTONI RAMIS SALVÀ, professors titulars del Departament de Sanitat i d’Anatomia Animals de la Facultat de Veterinària de la Universitat Autònoma de Barcelona i investigadors adscrits al Institut de Recerca i Tecnologia Agroalimentàries-Programa de Sanitat Animal (IRTA-CReSA), Certifiquen: Que la memòria titulada “Unravelling the pathobiological diversity of highly pathogenic avian influenza in birds” presentada per Raúl Sánchez González per a l’obtenció del grau de Doctor, s’ha realitzat sota la seva direcció i supervisió i, considerant-la acabada, n’autoritzen la seva presentació per tal de ser avaluada per la comissió corresponent. I per tal que consti als efectes oportuns, signen el present certificat a Bellaterra, Dra. Natàlia Majó i Masferrer Dr. Antoni Ramis Salvà Raúl Sánchez González Directora Director Doctorand Els estudis de doctorat de Raúl Sánchez González han estat finançats per una beca d’Ajuts per a la contractació de personal investigador novell (FI-DGR 2016), concedida per l’Agència de Gestió d’Ajudes Universitàries i de Recerca (AGAUR) de la Generalitat de Catalunya, referència 2016FI_B 00749. Aquest treball ha estat finançat pel projecte RTA2015-00088-C03-03 del Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Abbreviations.…..……………………………………………………………………………....I Summary/Resum................................................................................................................................................................V PART I: GENERAL INTRODUCTION AND OBJECTIVES................................1 CHAPTER 1: GENERAL INTRODUCTION...........................................................................3 1. AVIAN INFLUENZA INFECTION………………………………………...…...5 1.1. HISTORY……………………………………………………………………...….5 1.2. ETIOLOGY…………………………………………………………………...….9 1.2.1. Classification and nomenclature……………………………………...…....9 1.2.2. Viral structure and protein functions…………………………………...…10 1.2.3. Virus replication cycle…………………………………………………....13 1.2.4. Antigenic evolution………………………………………………........…15 1.2.5. Viral pathotypes…………………………………………………….........16 1.3. EPIDEMIOLOGY………………………………………………………….....…18 1.3.1. Host range…………………………………………………………........18 1.3.2. Reservoirs…………………………………………………………...….20 1.3.3. Introduction and dissemination……………………………………….....22 1.4. PATHOBIOLOGY…………………………………………………………...….24 1.4.1. Pathogenesis………………………………………………….………....25 1.4.2. Clinical presentation …………………………………………….….…...27 1.4.3. Gross and microscopic lesions…………………………………………...36 1.4.4. Determinants of infection……………………………………………......37 1.4.4.1. Viral factors…………………………………………..……….37 1.4.4.2. Host factors……………………………………………..….....39 1.5. PREVENTION, DIAGNOSIS AND CONTROL………………………..…...….41 CHAPTER 2: HYPOTHESIS AND OBJECTIVES………….……………….....47 PART II: STUDIES..............................................................................................................................................51 CHAPTER 3. STUDY I: Genetic characterization of the highly pathogenic avian influenza A H5N8 (clade 2.3.4.4B) virus isolated from domestic waterfowl in Catalonia, Spain, during the 2016- 2017 European epidemics……………………………………………..…...............……….…......53 3.1. INTRODUCTION………………………………………………………...............55 3.2. MATERIALS AND METHODS…………………………………………..…......56 3.3 RESULTS……………………………………………………………………...…57 3.4. DISCUSSION………………………………………………………………..…..63 CHAPTER 4. STUDY II: Pathobiology of the highly pathogenic avian influenza viruses H7N1 and H5N8 in different chicken breeds and role of Mx 2032 G/A polymorphism in infection outcome…………………………………………………………………………..……....…...67 4.1. INTRODUCTION…………………………………………………….……...…69 4.2. MATERIALS AND METHODS…………………………………………..…......70 4.3 RESULTS………………………………………………………………….............75 4.4. DISCUSSION…………………………………………………….…………........85 CHAPTER 5. STUDY III: Experimental infection of domestic geese (Anser anser var. domesticus) with highly pathogenic avian influenza viruses H5N8 and H7N1 reveals large differences in their virulence and potential transmissibility…………………………..................................….…............….91 5.1. INTRODUCTION…………………………………………………………...…93 5.2. MATERIALS AND METHODS………………………………………….…….94 5.3 RESULTS…………………………………………………………………….......98 5.4. DISCUSSION………………………………………………………………….106 CHAPTER 6. STUDY IV: Experimental inoculation of local and urban pigeons (Columba livia var.domestica) with a classical (H7N1) and a Gs/GD H5 (H5N8) highly pathogenic avian influenza virus ……………………………………………………………………………...................................113 6.1. INTRODUCTION……………………………………………………………..115 6.2. MATERIALS AND METHODS……………………………………..……..…..116 6.3 RESULTS…………………………………………………………..…………....119 6.4. DISCUSSION……………………………………………………………….......123 PART III: GENERAL DISCUSSION AND CONCLUSIONS.............................129 CHAPTER 7: GENERAL DISCUSSION…….............…………………....................131 CHAPTER 8: CONCLUSIONS…………………...............…………………….…....145 SUPPLEMENTARY MATERIAL………………………………………………………151 REFERENCES…………………………………………………………………………..161 ABBREVIATIONS AI Avian influenza AIV(s) Avian influenza virus (viruses) BSL-3 Biosecurity level 3 C-ELISA Competitive enzyme-linked immunosorbent assay CNS Central nervous system CReSA Centre de Recerca en Sanitat Animal cRNA Complementary RNA CS Cloacal swabs Ct Cycle threshold DNA Deoxyribonucleic acid Dpi Days post-inoculation DPPA Densely Populated Poultry Areas EID50 50% egg infective dose ELD50 50% embryo lethal dose ELISA Enzyme-linked immunosorbent assay EU European Union FAO Food and Agriculture Organization of the United Nations FP Feather pulp HA Hemagglutinin HE Hematoxylin/eosin HPAI Highly pathogenic avian influenza HPAIV(s) Highly pathogenic avian influenza virus (viruses) HPNAI Highly pathogenic notifiable avian influenza Hpi Hours post-inoculation IA Influenza A IAV(s) Influenza A virus (viruses) Ig Immunoglobulin IHC Immunohistochemistry IRTA Institut de Recerca i Tecnologia Agroalimentàries IVPI Intravenous pathogenicity index LPAI Low pathogenic avian influenza LPAIV(s) Low pathogenic avian influenza virus (viruses) I ABBREVIATIONS LPM Live poultry market LPNAI Low pathogenic notifiable avian influenza MBCs Multibasic cleavage site MCs Monobasic cleavage site MDT Mean death time mRNA Messenger RNA M1 Matrix protein M2 Membrane ion channel protein NA Neuraminidase NAI Notifiable avian influenza NDV Newcastle disease virus NSL Nuclear localization signal NP Nucleoprotein NS1 Nonstructural protein 1 NS2/NEP Nonstructural protein 2/Nuclear export proteins OIE World Organization for Animal Health OS Oropharyngeal swabs PA Polymerase acidic protein PBS Phosphate-buffered saline PB1 Polymerase basic protein 1 PB2 Polymerase basic protein 2 qRT-PCR Quantitative reverse transcription polymerase chain reaction RNA ribonucleic acid RNP Ribonucleoprotein SA Sialic aicd receptors SEM Standard error mean SPF Specific pathogen free vRNA Genomic viral RNA VSV Vesicular stomatitis virus II SUMMARY Avian influenza (AI) is considered one of the most important viral diseases affecting the poultry industry and a continuous threat to human population and wildlife. The majority of highly pathogenic avian influenza (HPAI) epidemics have affected land-based poultry, and classical lineages of HPAI viruses (HPAIVs) have been more sporadically isolated or rarely caused high mortality in aquatic poultry, wild birds and peridomestic avian species. However, the epidemiology and pathobiology of HPAI have radically changed since the emergence of Goose/Guangdong (Gs/GD) H5 lineage of HPAIVs. This lineage present unique biological characteristics among HPAIVs, including the capacity to infect and cause mortality in a broad range of domestic, captive and wild avian species. These demonstrate the large differences in infection outcome depending of the HPAIV isolate. Several studies show that the infection outcome is also influenced by host factors. Particularly, a broad variation in susceptibility to HPAIV infection exists among chicken breeds, suggesting that the genetic background of particular breeds confers a higher resistance to HPAIV infection. Usually, local chicken breeds have been considered more resistant to disease than commercial breeds due to lack of artificial selection towards