Unravelling the Pathobiological Diversity of Highly Pathogenic Avian Influenza in Birds

Unravelling the pathobiological diversity of highly pathogenic avian influenza in birds Raúl Sánchez González ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús <ul style="display: flex;"><li style="flex:1">establertes per la següent llicència Creative Commons: </li><li style="flex:1"><a href="/goto?url=http://cat.creativecommons.org/?page_id=184" target="_blank">http://cat.creativecommons.org/?page_id=184 </a></li></ul>ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: <a href="/goto?url=http://es.creativecommons.org/blog/licencias/" target="_blank">http://es.creativecommons.org/blog/licencias/ </a>WARNING. 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: <a href="/goto?url=https://creativecommons.org/licenses/?lang=en" target="_blank">https://creativecommons.org/licenses/?lang=en </a>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 LaDra.NATÀLIAMAJÓiMASFERRERielDr.ANTONIRAMISSALVÀ, professorstitularsdel Departamentde Sanitatid’Anatomia Animalsde la FacultatdeVeterinàriade la UniversitatAutònoma 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 Directora Dr. Antoni Ramis Salvà Director Raúl Sánchez González 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 ofthe highly pathogenic avian influenza virusesH7N1 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 (Anse r a nser 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 <ul style="display: flex;"><li style="flex:1">AI </li><li style="flex:1">Avian influenza </li></ul>AIV(s) BSL-3 C-ELISA CNS Avian influenza virus (viruses) Biosecurity level 3 Competitive enzyme-linked immunosorbent assay Central nervous system CReSA cRNA CS Centre de Recerca en Sanitat Animal Complementary RNA Cloacal swabs <ul style="display: flex;"><li style="flex:1">Ct </li><li style="flex:1">Cycle threshold </li></ul>DNA Dpi Deoxyribonucleic acid Days post-inoculation DPPA EID50 ELD50 ELISA EU Densely Populated Poultry Areas 50% egg infective dose 50% embryo lethal dose Enzyme-linked immunosorbent assay European Union FAO FP Food and Agriculture Organization of the United Nations Feather pulp <ul style="display: flex;"><li style="flex:1">HA </li><li style="flex:1">Hemagglutinin </li></ul><ul style="display: flex;"><li style="flex:1">HE </li><li style="flex:1">Hematoxylin/eosin </li></ul>HPAI HPAIV(s) HPNAI Hpi Highly pathogenic avian influenza Highly pathogenic avian influenza virus (viruses) Highly pathogenic notifiable avian influenza Hours post-inoculation <ul style="display: flex;"><li style="flex:1">IA </li><li style="flex:1">Influenza A </li></ul>IAV(s) Ig Influenza A virus (viruses) Immunoglobulin <ul style="display: flex;"><li style="flex:1">IHC </li><li style="flex:1">Immunohistochemistry </li></ul>IRTA IVPI LPAI LPAIV(s) Institut de Recerca i Tecnologia Agroalimentàries Intravenous pathogenicity index Low pathogenic avian influenza Low pathogenic avian influenza virus (viruses) IABBREVIATIONS LPM LPNAI MBCs MCs MDT mRNA M1 Live poultry market Low pathogenic notifiable avian influenza Multibasic cleavage site Monobasic cleavage site Mean death time Messenger RNA Matrix protein <ul style="display: flex;"><li style="flex:1">M2 </li><li style="flex:1">Membrane ion channel protein </li></ul><ul style="display: flex;"><li style="flex:1">Neuraminidase </li><li style="flex:1">NA </li></ul><ul style="display: flex;"><li style="flex:1">NAI </li><li style="flex:1">Notifiable avian influenza </li></ul>Newcastle disease virus Nuclear localization signal Nucleoprotein NDV NSL NP <ul style="display: flex;"><li style="flex:1">NS1 </li><li style="flex:1">Nonstructural protein 1 </li></ul>Nonstructural protein 2/Nuclear export proteins World Organization for Animal Health Oropharyngeal swabs NS2/NEP OIE OS <ul style="display: flex;"><li style="flex:1">PA </li><li style="flex:1">Polymerase acidic protein </li></ul>Phosphate-buffered saline Polymerase basic protein 1 Polymerase basic protein 2 Quantitative reverse transcription polymerase chain reaction ribonucleic acid PBS PB1 PB2 qRT-PCR RNA RNP SA Ribonucleoprotein Sialic aicd receptors SEM SPF Standard error mean Specific pathogen free vRNA VSV Genomic viral RNA Vesicular stomatitis virus II SUMMARY Avianinfluenza(AI)is consideredoneofthemostimportantviraldiseasesaffectingthepoultryindustry 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 production-related genes, which could be negatively associated with resistance to pathogens. Todate, a directcomparisonofthe pathobiology ofclassicalandGs/GD H5 HPAIVsinavian species belonging to distinct taxonomic groups is lacking. The variation in susceptibility to HPAIV infection amongchicken breedshasnotbeen studiedin detailin Europe, aswell astheexistenceofbreed-related differences in susceptibility to HPAIVs in minor and peridomestic avian species. Consequently, in the present dissertation we systematically evaluated the differential pathobiological features of a HPAIV belonging to a classical lineage (H7N1 isolated in Italy in 1999) and a HPAIV of Gs/GD H5 lineage (H5N8 isolated in Spain in 2017) in different breeds of chickens (Gallus gallu s d omesticus), domestic geese (Anser anser var. domestica) and pigeons (Columba livia var. domestica). In Study I, the Gs/GD H5N8 HPAIV isolated in Spain in 2017 was genetically characterized. The pathobiological properties of Gs/GD H5N8 HPAIV were then compared side by side with the classical H7N1 HPAIV in three experimental infections (Studies II, III and IV). We evaluated the differences in clinical presentation, gross and microscopic lesions, distribution of viral antigen in tissues (IHC techniques), viral shedding (qRT-PCR technique) and seroconversion (cELISA) between both HPAIVs after intranasal inoculation in chickens (II), geese (III) and pigeons (IV). In Study II, the genotype and allele frequency of a single nucleotide polymorphism (SNP) at position 2032 of chicken Mx gene and their association with susceptibility to HPAIVs were also determined. V

Unravelling the Pathobiological Diversity of Highly Pathogenic Avian Influenza in Birds

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