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

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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

AI

Avian influenza

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

Ct

Cycle threshold

DNA Dpi
Deoxyribonucleic acid Days post-inoculation
DPPA

EID₅₀

ELD₅₀ELISA 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

HA

Hemagglutinin

HE

Hematoxylin/eosin

HPAI HPAIV(s) HPNAI Hpi
Highly pathogenic avian influenza Highly pathogenic avian influenza virus (viruses) Highly pathogenic notifiable avian influenza Hours post-inoculation

IA

Influenza A

IAV(s) Ig
Influenza A virus (viruses) Immunoglobulin

IHC

Immunohistochemistry

IRTA IVPI LPAI LPAIV(s)

Institut de Recerca i Tecnologia Agroalimentàries

Intravenous pathogenicity index Low pathogenic avian influenza Low pathogenic avian influenza virus (viruses)

I

ABBREVIATIONS

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

M2

Membrane ion channel protein

Neuraminidase

NA

NAI

Notifiable avian influenza

Newcastle disease virus Nuclear localization signal Nucleoprotein
NDV NSL NP

NS1

Nonstructural protein 1

Nonstructural protein 2/Nuclear export proteins World Organization for Animal Health Oropharyngeal swabs
NS2/NEP OIE OS

PA

Polymerase acidic protein

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