Identification et caractérisation des facteurs cellulaires requis pour l’infection du virus Zika et du virus de la Dengue

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Identification et caractérisation des facteurs cellulaires

requis pour l’infection du virus Zika et du virus de la

Dengue

Athena Labeau

To cite this version:

Athena Labeau. Identification et caractérisation des facteurs cellulaires requis pour l’infection du virus Zika et du virus de la Dengue. Médecine humaine et pathologie. Université de Paris, 2019. Français. �NNT : 2019UNIP7060�. �tel-03100476�

Université de Paris

Ecole doctorale Hématologie Oncogénèse et Biothérapies (HOB) ED561

Institut de Recherche Saint-Louis

Laboratoire « Biologie cellulaire des infections virales »

INSERM U944-CNRS UMR7212

Identification and characterization of cellular factors

required for Zika virus and Dengue virus infection

Par Athéna LABEAU

Thèse de doctorat de Virologie fondamentale

Dirigée par le Dr Ali AMARA

Présentée et soutenue publiquement le 16 Décembre 2019

Devant le jury composé de :

Pr Constance DELAUGERRE

Présidente

Dr Yves ROUILLE

Rapporteur

Dr Damien VITOUR

Rapporteur

Dr Nathalie PARDIGON

Examinatrice

Dr Timothée BRUEL

Examinateur

Dr Laurent MEERTENS

Examinateur

Remerciements

En premier lieu, je souhaite exprimer toute ma gratitude à mon directeur de thèse, le Dr Ali Amara, pour la confiance qu’il m’a accordé en me donnant l’opportunité de rejoindre son équipe. Malgré, nos quelques divergences d’opinions, je te remercie de tout cœur des enseignements que tu m’as apporté et qui me permettront de m’intégrer dans le monde professionnel. J’espère sincèrement que tu as pu, comme moi, sortir enrichi de nos échanges.

Je remercie mes deux rapporteurs le Dr Damien Vitour et le Dr Yves Rouillé pour leur investissement dans la relecture de mon manuscrit. Je remercie également les autres membres du jury qui vont évaluer mon travail. Je suis profondément reconnaissante à la présidente de mon jury, le Pr Constance Delaugerre, ainsi qu’à mes examinateurs le Dr Nathalie Pardigon, le Dr Timothée Bruel et le Dr Laurent Meertens.

A toi Laurent, qui m’a encadrée et supportée durant ces quatre années du Master au Doctorat, je n’ai pas de mots pour te dire à quel point je te suis reconnaissante. Croiser ton chemin a fait de moi, une meilleure scientifique. Merci de tout cœur pour tous ces moments partagés : nos débats interminables sur le sens de la vie, nos fous rires, tes blagues géniales, ton écoute et tes précieux conseils dans mes choix professionnels et personnels. Un grand merci également pour l’énorme soutien que tu m’as apporté durant ma grossesse. Je te suis profondément redevable pour l’aide que tu m’as apportée dans la correction de la discussion de mon manuscrit. Il y a des personnes qui nous marquent et nous font grandir, tu es l’une de ces personnes pour moi. Je te souhaite le meilleur et la réussite dans tes projets futurs.

Evidemment, je remercie également les autres membres de l’équipe. J’ai une pensée particulière pour les deux nouveaux doctorants Alexis et Vasiliya, à qui je souhaite une belle thèse. Comme on dit en créole « tchimbé rèd, pa moli » : le travail et les efforts payent toujours, alors persévérez. Merci à Alexis et Sylvain pour avoir été les nourrices de Cassian pour que j’avance dans mon projet. Je te souhaite, Sylvain, une merveilleuse expérience à Miami. Un merci à toi aussi Sarah, pour m’avoir aidée et conseillée pour la suite de cette aventure. Une pensée également pour toi, Lucie et merci pour ton aide. Nos discussions de « geek » me manqueront. Merci aux anciens membres de l’équipe. A Lamine, avec qui j’ai pu apprendre beaucoup scientifiquement comme personnellement. Nos longues discussions en pièce de culture cellulaire me manqueront. A Claudia, pour sa joie de vivre, ses conseils et son écoute. Je te souhaite une très grande réussite au Canada. Merci également à Estelle et Stéphane pour les thés et les fous rires partagés. Un merci également à Ophélie, pour ses conseils qui m’ont rassuré sur ma capacité d’être une bonne doctorante.

Un grand remerciement aux membres de mon comité de suivi de thèse : le Pr Olivier Schwartz, le Pr Alessia Zamborlini et le Dr Stéphane Emiliani, pour leur clairvoyance qui ont grandement aidé dans l’avancement de mon travail. Merci pour votre écoute et vos conseils qui ont améliorés mon expérience sociale au laboratoire.

Merci à tous les membres de la plateforme technologique de l’IRSL pour leur expertise et leur gentillesse : Niclas, Sophie, Christelle un grand merci pour tout !

Je n’oublie pas toutes les personnes que j’ai croisées au sein de l’Unité 944. A toutes les scientifiques et mamans, merci pour vos précieux conseils qui m’ont aidé à bien gérer ma thèse et ma grossesse. Je vous dis merci Alessia, Adeline, Joëlle, Florence et Amandine. Merci aux doctorants : Antoine, Etienne, Zach, Anastasia et Charlotte qui m’ont permis de prendre du recul en partageant leur expérience avec moi et également d'avoir

supporté mes jérémiades. Vous rencontrer a été un des moments que je retiendrai de ces années. Une pensée pour Pierre, sans qui les journées au laboratoire auraient été moins joyeuses. Merci pour ton humour et ta participation à approfondir ma culture vidéographique. Merci aux membres de l’équipe SAIB, pour les moments qu’ils ont échangés avec moi.

Je remercie aussi l’ensemble de nos collaborateurs, dont les expertises dans leurs domaines respectifs nous ont permis de réaliser ces travaux fructueux sur Axl et DPM.

Je remercie également tous les membres de l’école doctorale HOB, pour leur gentillesse ainsi que la gestion administrative de mon dossier durant ma grossesse et pour le reste de ma thèse. Un grand merci à Mme Deborah Depost ainsi qu’à Mme Aurélie Butelle, pour l’astuce de l’écharpe pour les premiers pas de Cassian.

Merci aux membres de l’ADELIH, avec qui j’ai pu organiser la troisième édition du congrès de l’association. Le temps partagé avec vous, m’a permis de révéler des capacités insoupçonnées d’organisation.

Je remercie mes amis de Guyane, Ruthly, Robinson et Frédéric, qui malgré la distance, m’ont encouragé et remonté le moral. Merci à toi aussi, Grégoire pour la relecture et la correction de mon manuscrit. J’espère qu’en le lisant tu as pu découvrir cet univers dont je te parlais tant. Je t’attends impatiemment pour ma revanche aux colons de Catan.

Merci à la collectivité territoriale de Guyane, pour le soutien financier qu’elle m’a apporté, sans oublier tous mes professeurs de Licence en Guyane qui ont cru en mes capacités et qui sont à l’origine de la scientifique que je suis devenue. Mme Hotin, Mme Prévôt, Mme Martial, Mr Robinson et Mr Bereau, je vous remercie du fond du coeur ! Vous façonnez la Guyane de demain.

Merci à ma famille pour leur confiance et leur soutien, grâce auxquels j’ai pu vivre ma passion. Merci pour votre aide, morale comme financière. Merci à ma maman et à Cédric, pour votre écoute, vos conseils, vos encouragements, vos relectures de mails et d’avoir su canaliser la pile électrique que je peux parfois être. Merci également à mes deux autres parents, mon père et « tatie » Annie pour leur aide dans mon installation en France et leurs encouragements. Un merci particulier à mon petit frère Rayman pour son écoute.

Je remercie aussi ma nouvelle famille pour tout le soutien que vous m’avez apporté. Même si vous ne compreniez rien à la virologie, vous n’avez cessé de m’aider avec les atouts que vous possédez. Merci à toi Delphine, chère belle-sœur, pour la correction de l’anglais de mon manuscrit de thèse, et à toi Valérie, ma chère belle-mère pour le coaching musclé de mon oral de thèse.

Pour finir, je dédie ce travail aux deux hommes de ma vie qui ont vécu cette thèse avec moi, mon fils Cassian et mon compagnon Julien. A Cassian, j'espère que lorsque tu seras en âge de le comprendre, tu seras fier de ta maman. Sache que le mérite te revient aussi car tu as su être un bébé sage et adorable pour que je finisse mon doctorat. Tu es la lumière qui m’a donné la force de me surpasser pour ces dernières années. A Julien, tu m’as accompagné dans cet univers qui t’était totalement inconnu. Avec brio, tu as su m’écouter et me conseiller. Merci pour la relecture de mes mails et l’habilité dont tu faisais preuve pour les corrections. Merci de m’avoir soutenu durant ma rédaction, pour le photoshoping de mes figures et pour la préparation de mon oral. La vi-a a pa roun bol toloman, mais ensemble nous y sommes arrivés.

1

RESUMÉ

Les virus de la Dengue (DENV) et Zika (ZIKV) sont deux virus émergents transmis par des moustiques et sont responsables de pathologies sévères chez l’homme. Il n’y a actuellement aucun vaccin efficace, ni traitements antiviraux disponibles contre ces arbovirus. DENV et ZIKV sont des parasites intracellulaires obligatoires qui dépendent entièrement de leur cellule hôte pour se multiplier. Cette dépendance constitue un potentiel talon d’Achille qui pourrait être exploité dans le développement de nouvelles stratégies thérapeutiques. L’objectif général de mon travail de thèse a été de comprendre les mécanismes par lesquels DENV et ZIKV exploitent les fonctions cellulaires à leur avantage, et d’identifier des facteurs de l’hôte requis à l’initiation de leur cycle infectieux.

ZIKV a un tropisme cellulaire pour le cerveau et est responsable de troubles neurologiques sévères tels que les microcéphalies congénitales chez le fœtus. La première partie de mon travail a été de caractériser le rôle du récepteur Axl dans le neurotropisme de ZIKV. Axl est un récepteur à la phosphatidylsérine appartenant à la famille des récepteurs TAM, qui est impliqué dans la reconnaissance et l’élimination des cellules apoptotiques par phagocytose. Ce travail de thèse a montré qu’Axl est abondamment exprimé par les cellules microgliales, les cellules gliales radiales et les astrocytes dans le cerveau en développement de fœtus et est important pour l’infection de ces cellules par ZIKV. Nous décrivons deux rôles distincts joués par Axl durant l’infection par ZIKV. Tout d’abord, Axl, par l’intermédiaire de son ligand Gas6, permet l’attachement et l’endocytose des particules virales dans les cellules gliales. Dans un deuxième temps et simultanément à l’entrée virale, les complexes Gas6-ZIKV agissent comme des « super » agonistes et phosphorylent le domaine intracellulaire à tyrosine kinase d’Axl, ce qui déclenche des cascades de transduction du signal aboutissant à l’inhibition de la réponse immunitaire innée et à une réplication virale optimale. En outre, nous avons identifié deux antagonistes d’Axl, le MYD1 et le R428, et avons montré leurs prometteuses propriétés antivirales in vitro.

La deuxième partie de mon travail a consisté à identifier de façon systématique les gènes de la cellule hôte qui sont importants pour le cycle infectieux de DENV. A l’aide d’un crible CRISPR-Cas9 à l’échelle du génome, nous avons identifié les protéines DPM1 et DPM3 comme de nouveaux facteurs de dépendance de DENV. DPM1 et DPM3 sont deux sous-unités du complexe dolichol-phosphate-mannose synthase (DPMS), localisé à la membrane du réticulum endoplasmique. Ce complexe catalyse la synthèse du dolichol-phosphate-mannose, qui fournit le mannose requis pour les différentes voies de glycosylation des protéines. A l’aide de cellules DPM knockout, nous avons montré que le complexe DPMS facilite l’infection des quatre sérotypes de DENV mais aussi de ZIKV ainsi que d’autres flavivirus apparentés tel que le virus de la fièvre jaune. A l’aide de mutants catalytiques de DPM1, nous avons pu décrire que l’activité catalytique de DPMS est cruciale pour l’infection par DENV. Nous avons montré que le complexe DPMS accomplissait différentes fonctions au cours du cycle infectieux de DENV. Ce complexe est nécessaire au mécanisme d’amplification du génome viral et, est également requis pour la N-glycosylation des protéines virales structurales, qui permet ainsi leur repliement correct.

En conclusion, ces travaux ont contribué à améliorer notre compréhension des mécanismes d’entrée et de réplication de deux arbovirus majeurs, que sont ZIKV et DENV. Nos études ont identifié Axl et le complexe DPMS comme des facteurs cellulaires importants pour le cycle infectieux de ZIKV et DENV et suggèrent que ces molécules pourraient constituer de nouvelles cibles pour une intervention antivirale.

Mots clefs : Axl, Gas6, neurotropisme du virus Zika, cellules gliales, Dengue, crible

2

SUMMARY

Dengue virus (DENV) and Zika virus (ZIKV) are two emerging viruses transmitted to humans by mosquitoes and are responsible for severe pathologies. There is currently no efficient vaccine neither antiviral treatment available against these arboviruses. DENV and ZIKV are fully dependent on the host cell for their multiplication. This dependence creates a “Achille’s heel” that may be exploited to develop new approaches to treat these viral infections. The general objective of my PhD is to understand the mechanisms by which DENV and ZIKV exploit the cellular functions for their advantage and to identify the host factors required for the initiation of their viral life cycle.

ZIKV displays a cellular tropism for the brain and is responsible for severe neurological disorders such as congenital microcephaly in the fetus. The first part of my work was to characterize the role of the Axl receptor in the ZIKV neurotropism. Axl is a phosphatidylserine receptor belonging to the TAM receptor family, which is involved in the recognition and the removal of the apoptotic cells by phagocytosis. This work showed that Axl is highly expressed by microglial cells, radial cells and astrocytes in developing brain of fetuses and is important for ZIKV infection of these cells. We described two distinct roles played by Axl during ZIKV infection. First Axl, through its ligand Gas6, promotes the adsorption and the endocytosis of viral particles into glial cells. Second and simultaneously to the viral entry, the Gas6-ZIKV complexes act a ‘super agonist” that phosphorylate Axl tyrosine kinase domain to trigger signaling cascades that lead to the inhibition of the innate immune response and optimal viral replication. Furthermore, we identified two antagonists of Axl, the MYD1 and the R428, and showed their promising antiviral properties in vitro.

The second part of my work aims to identify the cellular genes required for DENV infection. Using a genome-wide CRISPR-Cas9 screen, we identified the DPM1 and DPM3 proteins as new DENV host dependency factors. DPM1 and DPM3 are two subunits of the phosphate-mannose synthase (DPMS) complex. This latter catalyzes the dolichol-phosphate-mannose synthesis, which provides the mannose required for the different glycosylation pathways in the ER lumen. Using DPM knockout cells, we showed that the DPMS complex facilitates the infection of the four DENV serotypes as well as other flaviviruses such as ZIKV and yellow fever virus (YFV). Using DPM1 mutants, we found that the catalytic activity of DPMS is crucial for DENV infection. We showed that DPMS complex plays several functions during the DENV life cycle. This complex is necessary for the replication of the viral genome, and is also required for the N-glycosylation of structural viral proteins, which allows their correct folding.

In conclusion, this PhD work provides new insights for our understanding of the entry and replication mechanisms of two major arboviruses. These studies identified Axl and the DPMS complex as important cellular factors required for ZIKV and DENV life cycle and suggest that targeting these molecules may represent new strategies to combat DENV and ZIKV infection.

Keywords: Axl, Gas6, ZIKV neurotropism, glial cells, Dengue, CRISPR-Cas9 screen,

3

INDEX

II. ZIKV AND DENV: TWO PATHOGENIC FLAVIVIRUSES 14

1. ZIKA VIRUS 14

a. History and emergence of Zika Virus 14

b. Transmission 15

c. Epidemiology 16

d. Pathogenesis 17

e. Treatment and vaccines 18

2. DENGUE VIRUS 20

a. History and emergence of Dengue Virus 20

b. Transmission 20

c. Epidemiology 22

d. Pathogenesis 23

e. Treatment and vaccines 25

III. FLAVIVIRUS GENOME AND VIRAL PARTICLE ORGANIZATION 26

1. GENOME ORGANIZATION 26 2. NON-STRUCTURAL (NS) PROTEINS 29 a. NS1 29 b. NS2A 30 c. NS2B 31 d. NS3 31 e. NS4A 32 f. NS4B 33 g. NS5 33 3. STRUCTURAL PROTEINS 35 a. Capsid protein 35 b. Membrane protein 37 c. Envelope protein 37

4. VIRAL PARTICLES ORGANIZATION 39

a. Maturation process 39

b. Mature viral particles 40

c. Immature viral particles 42

d. Partially mature viral particles 43

5. FLAVIVIRUS LIFE CYCLE 44

IV. FLAVIVIRUS ENTRY RECEPTORS 46

4

2. THE TIM AND TAM RECEPTORS 47

V. THE AXL RECEPTOR: STRUCTURE, FUNCTIONS AND IMPLICATION IN ZIKV INFECTION 49

1. EXPRESSION AND STRUCTURE 49

2. AXL AND TAM RECEPTORS SIGNALING 51

3. ROLE OF AXL RECEPTOR DURING ZIKV INFECTION 53

VI. IDENTIFICATION OF NEW HOST DEPENDENCY FACTORS BY GENOME-WIDE CRISPR-CAS9 SCREENS 55

1. CRISPR-CAS SYSTEM 55

2. GENOME-WIDE POOLED SGRNA SCREENING TO IDENTIFY HOST FACTORS REQUIRED FOR FLAVIVIRUS

INFECTION 56

a. sgRNA libraries 58

b. Flavivirus CRISPR-Cas9 screens 58

VII. OBJECTIVES 62

RESULTS 63

I. AXL MEDIATES ZIKA VIRUS ENTRY IN HUMAN GLIAL CELLS AND MODULATES INNATE IMMUNE

RESPONSES (CELL REPORTS,2017) 64

II. A GENOME-WIDE CRISPR-CAS9 SCREEN IDENTIFIES THE DOLICHOL-PHOSPHATE MANNOSE SYNTHASE COMPLEX AS A HOST DEPENDENCY FACTOR FOR DENGUE VIRUS INFECTION (UNDER REVIEW) 65

DISCUSSION AND PERSPECTIVES 66

I. EFFECTIVE CONTRIBUTION OF AXL IN ZIKV ENTRY, IMMUNE EVASION AND PATHOGENESIS 67

1. THE ROLE OF AXL IN ZIKV ENTRY PROCESSES 67

2. THE ROLE OF AXL IN THE IMMUNE RESPONSE REGULATION 69

3. THE ROLE OF AXL IN ZIKV PATHOGENESIS 70

4. CONCLUDING REMARKS 72

II. GLYCOSYLATION PROCESSES IN THE DENV LIFE CYCLE 73

1. IDENTIFICATION OF CELLULAR FACTORS REQUIRED FOR DENV INFECTION USING A GENOME-WIDE

CRISPR-CAS9 SCREEN 73

2. THE N-GLYCOSYLATION FUNCTIONS OF DPMS COMPLEX IS REQUIRED FOR DENV INFECTION 75

3. ROLE OF N-GLYCOSYLATION IN THE STABILITY OF DENV PROTEINS 77 4. GPI-ANCHORED MOLECULES MEDIATE DENV ENTRY IN HAP1 CELLS 80

5. CONCLUDING REMARKS 81

REFERENCES 83

5

FIGURES INDEX

Figure 1. Four genera of Flaviviridae family. ... 11

Figure 2. Flavivirus genus classification. ... 13

Figure 3. Geographic regions where ZIKV is enzootic/endemic and has caused epidemics. 15 Figure 4. Reported forms of ZIKV transmission. ... 16

Figure 5. Areas of ZIKV introduction. ... 17

Figure 6. Transmission cycles of DENV. ... 22

Figure 7. Global Dengue Risk. ... 23

Figure 8. Flavivirus genome organization and polyprotein processing. ... 27

Figure 9. Flavivirus genome 5'-3' long distance interaction. ... 29

Figure 10. Flavivirus replication complex... 35

Figure 11. Structure and conformations of the ZIKV E protein ... 38

Figure 12. The maturation pathways for flaviviruses. ... 40

Figure 13. Structure of mature flavivirus particles. ... 41

Figure 14. Conformation of mature DENV particles change depending on temperature variations. ... 42

Figure 15. Structure of immature flavivirus particles. ... 43

Figure 16. Structure of partially mature flavivirus particles. ... 44

Figure 17. The life cycle of flaviviruses. ... 45

Figure 18. Apoptotic mimicry: a mechanism to promote virus infection ... 48

Figure 19. TAM receptors structure with their ligands ... 50

Figure 20. Inhibition of the innate immune response by Axl receptor ... 53

Figure 21. CRISPR: a prokarytic adaptative immune mechanism ... 55

Figure 22. A pooled approach for genetic screening in eukaryotic cells using a lentiviral CRISPR-Cas9 system. ... 57

Figure 23. Lipid-linked oligosaccharide synthesis in WT cells versus DPM1 or DPM3 KO cells ... 76

Figure 24. N-glycan, a sensor of the ER quality control of the proteins. ... 78

Figure 25. Schematic representation of Man5GlcNAc2 glycoforms. ... 79

Figure 26. A role of the DPMS complex in DENV entry in HAP1 cells ... 81

6

ABBREVIATIONS LIST

AC Apoptotic cell

ADE Antibody-Dependent Enhancement

Ala Alanine

ALG Asparagine Linked Glycosylation

APCs Antigen Presenting Cells

Arg Arginine

Asn Asparagine

ATP Adenosine Triphosphate

C Capsid

CDG Congenital Disorders of Glycosylation

cGAS GMP-AMP Synthase

CHIKV Chikungunya virus

CNS Central Nervous System

CNX Calnexin

CRD Carbohydrate Recognition Domain

CRT Calreticulin

CRISPR-Cas9 Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9

CS Cyclization Sequence

CTLR C-Type Lectin Receptor

Cryo-EM Cryo-Electron Microscopy

DCs Dendritic Cells

DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin

DENV Dengue virus

DF Dengue Fever

DNA Deoxyribonucleic Acid

Dol-P Dolichol-Phosphate

DPG Dolichol-Phosphate-Glucose

DPM Dolichol-Dhosphate-Mannose

DPMS Dolichol-Dhosphate-Mannose Synthase

dsRNA double-stranded RNA

DSS Dengue Shock Syndrome

E Envelope

EBOV Ebola virus

7

EGF Epidermal Growth Factor

EMC ER Membrane protein Complex

ER Endoplasmic Reticulum

ERAD Endoplasmic-Reticulum-Associated protein Degradation

ERman1 ER alpha-mannosidase 1

FcγR Fcγ Receptor

GAG Glycosaminoglycans

Gas6 Growth Arrest Specific gene 6

GBS Guillain-Barre Syndrome

GDP Guanosine Diphosphate

GlcNAc β-1,2 N-Acetylglucosamine

Glu Glutamic acid

GPI Glycosylphosphatidylinositol

HAV Hepatitis A virus

HCV Hepatitis C virus

HDF Host Dependency Factor

HIV Human Immunodeficiency virus

hNPCs human Neural Progenitor Cells

HS Heparan Sulfate

IAV Influenza A virus

IFN Interferon

IFNAR1 Interferon Alpha/beta Receptor 1

Ig Immunoglobulin

IL Interleukin

IRF Interferon Regulatory Factor

ISG Interferon Stimulated Genes

ISRE Interferon-Stimulated Response Element

JEV Japanese Encephalitis virus

KO Knock Out

LDs Lipid Droplets

LLO Lipid-Linked Oligosaccharides

LNPs Lipid Nanoparticles

L-SIGN Liver/lymph node-Specific ICAM-3 Grabbing Non integrin

Lys Lysine

M Membrane

MTase Methyltransferase

8

NES Nuclear Export Sequence

NF-κB Nuclear Factor-kappa B

NGS Next-Generation Sequencing

NHPs Nonhuman Primates

NKV No Known Vector

NLS Nuclear Localization Sequence

NPCs Neural Progenitor Cells

NS Non-Structural

ORF Open Reading Frame

OST Oligosaccharyltransferase

PAMPs Pathogen Associated Molecular Patterns

PCR Polymerase Chain Reaction

Phe Phenylalanine

PI Phosphatidylinositol

PIG Phosphatidylinositol Glycan

PKR Protein Kinase R

PMM Phosphomannomutase

POMT Protein O-Mannosyltransferase

PTK Protein-Tyrosine Kinase

prM pre-Membrane

ProS Protein S

PtdSer Phosphatidylserine

RCS Repeat Cyclization Sequence

RdRp RNA-dependant RNA polymerase

RNA Ribonucleic Acid

RTK Receptor Tyrosine Kinase

SC Sertolli Cell

Ser Serine

sgRNA Single guide RNA

SHBG Sex Hormone-Binding Globulin

SL Stem-Loop

SOCS Suppressor Of Cytokine Signaling

ssRNA single-stranded RNA

STAT Signal Transducer and Activator of Transducer

STING Stimulator of Interferon Gene

TAM Tryo3, Axl, Mer

9 TBK1 TANK-Binding Kinase 1

TGN Trans-Golgi network

Thr Threonin

TIM T cell, Immunoglobulin domain, and Mucin domain

TLR Toll-Like Receptor

TNF Tumor Necrosis Factor

Trp Tryptophan

Tyr Tyrosine

UGGT UDP-glucose:glycoprotein glucosyltransferase

UPR Unfolded Protein Response

UTRs Untranslated Regions

VACV Vaccinia virus

VLPs Virus-Like Particles

VPs Vesicle Packets

VR Variable Region

vRNA Viral RNA

WNV West Nile virus

WT Wild-Type

YFV Yellow Fever virus

10

11

I. Flaviviruses

1. Overview

The genus Flavivirus belongs to the Flaviviridae family, which contains a large variety of viruses that share important similarities in genome structure, virion morphology and life cycle. This family consists of four genera which are differentiated by biological and antigenic properties: Flavivirus (from Latin “Flavus” means yellow), Hepacivirus (from Greek “Hepar”

meaning liver), Pestivirus (from Latin “Pestis” meaning plague) and the newly proposed genus Pegivirus (“Pe” from persistent and “g” as histological reference to the former names of the

human viruses) (Figure 1) 1,2_.

Figure 1. Four genera of Flaviviridae family.

Flaviviridae family is classified in four genera: Flavivirus, Hepacivirus, Pegivirus and Pestivirus. Phylogenetic tree

based on the analysis of aligned conserved motifs of the RNA dependent RNA polymerase Adapted from 3_.

The Flavivirus genus is the largest genus within the Flaviviridae family. It includes more than 70 enveloped arboviruses (or arthropod-borne viruses), transmitted to vertebrate hosts by ticks or mosquitoes. This genus contains viruses considered as emerging human pathogens such as viruses of Yellow Fever Virus (YFV), Japanese Encephalitis Virus (JEV), Tick-Borne Encephalitis Virus (TBEV), West Nile Virus (WNV), Dengue Virus (DENV) and Zika Virus (ZIKV). Except, for YFV and DENV, infected humans are not able to sustain the natural

12 cycle of transmission of flaviviruses and so are considered as incidental “dead-end hosts 4_.

Currently, flaviviruses are widespread in all continents and are endemic in many parts of the world due to the global distribution of their vectors.

2. Classification

Flaviviruses were first classified in 1974 as the genus Flavivirus with three others (Alphavirus, Pestivirus and Rubivirus) of the Togaviridae family. Based on several fundamental differences compared to the Togaviridae such as structural characteristics, replication strategy and gene sequence, in 1984 the creation of a new family was proposed, the Flaviviridae family, initially composed of the single genus Flavivirus 2_{. Moreover in 1988, another study has}

classified the sixty-eight recognized members contained in Flaviviridae family by cross-neutralisation assays and divided them into eight complexes, of which a quarter of the members could not be associated with any of these complexes 5_{. Later, the sequence}

comparison of the flavivirus non-structural 5 (NS5) gene allowed the classification of all the isolated viruses into three different clusters: mosquito-borne, tick-borne and no known vector (NKV) clusters 5–7_{. Furthermore, these clusters were subdivided in groups according to the}

transmission vector nature, the principal vertebrate host and the type of disease (Figure 2) 8_.

Mosquito-borne flavivirus can be assigned in two groups. The first composed of flaviviruses such as WNV, JEV, Murray Encephalitis Virus and Saint-Louis Encephalitis Virus (SLEV) are essentially transmitted by mosquitoes from the Culex genus with birds as preferential vertebrate hosts while the second composed of flaviviruses such as YFV, the four DENV serotypes and ZIKV are essentially transmitted by Aedes mosquitoes with primates and humans as vertebrate hosts 8,9_.

13

Figure 2. Flavivirus genus classification.

Phylogenetic tree based on the analysis of the flaviviruses NS5 gene sequence. Different subgroups are distinguished by vector association (tick, mosquitoes or known vector (NKV)), the host (mammalian or birds). Adapted from 8_.

Tick-borne flaviviruses also forms two distinct groups. The first with the seabirds as vertebrate hosts and the second, the tick-borne encephalitis complex viruses, associated primarily with rodents. The NKV flaviviruses forms three distinct groups. Two groups with bats as vertebrate hosts, one of which is closely related to mosquito-transmitted viruses and the other is more genetically distant and the third group with rodents as vertebrate hosts 8_.

14

II. ZIKV and DENV: two pathogenic flaviviruses

1. Zika Virus

a. History and emergence of Zika Virus

Zika Virus (ZIKV) was initially discovered on the African continent. It was first isolated in 1947 from the blood of a febrile sentinel rhesus monkey (no. 766) in the Zika Forest in Uganda during studies designed to identify the vector of sylvatic YFV 10_{. A few months later,}

another strain of ZIKV was isolated from Aedes africanus mosquitoes collected in the same area of the Zika forest. The first supposed case of human infection is controversial. The first report was from serum in 1954 of a 10-year-old Nigerian female in 1954 10_{. However, it has}

been suggested that the first case of confirmed human ZIKV infection occurred in Uganda in 1962-1963 11_{. Apart from the African continent, ZIKV was isolated for the first time from Aedes} aegypti mosquitoes in 1966 in Malaysia 12, then the first human infection was reported in 1977

in Central Java, Indonesia 13_.

Before 2007, despite its known existence, no case of epidemic ZIKV was ever reported. Only 14 human cases of ZIKV infection arose sporadically in Africa and Asia and were misdiagnosed 14,15_{. In 2007, the first known ZIKV epidemic occurred in the isolated islands}

of Yap, Federated States of Micronesia, located in the Western Pacific with 49 confirmed cases and 59 probable cases of ZIKV infection. However, the outbreak was reported as relatively small (approximately 5000 infections, approximately 75% of the population).

Until October 2013, no epidemic ZIKV transmission was reported. Then, other outbreaks occurred in French Polynesia, a South Pacific territory, where an estimated 11% of population were infected 16 _{while in Southeast Asia only sporadic transmission was occurring} 17_{. A major epidemic occurred in 2013/2014 involving all French Polynesian islands with more}

than 30 000 cases, some with neurologic complications 18_{. The virus spread to New Caledonia,}

the Cook Islands, Easter Island, and to the rest of the South Pacific 19_{. During the first months}

of 2015, the first native cases of ZIKV were reported in Brazil. However, the latest data suggest that the virus was introduced into Brazil as early as 2013 or 2014 20 _{from the Pacific, but the}

disease was not recognized until November 2015, when a major epidemic of neurologic symptoms in newborn babies occurred, with a second peak in April 2016 14,21_{. ZIKV has spread}

explosively across south and central Americas (Figure 3). Because of this quick spread and the alarming associated diseases, in February 2016, the World Health Organization declared ZIKV as “public health emergency of international concern” 22_{. However, after the fifth meeting}

15 of the Emergency Committee (EC) on ZIKV, microcephaly and other neurological disorders in 18 November 2016, the Director-General accepted the recommendations of the EC and declared the end of the Public Health Emergency of International Concern (PHEIC).

Figure 3. Geographic regions where ZIKV is enzootic/endemic and has caused epidemics. World map represents ZIKV circulation and the different outbreaks since virus isolation 23_.

b. Transmission

ZIKV has been recognized as an arthropod-borne virus (arbovirus). Since then, multiple confirmed modes of transmission have been reported.

Mosquito vectors primarily transmit ZIKV. In Africa, ZIKV circulates in a sylvatic transmission cycle involving nonhuman primates (NHPs) and arthropod vector mosquitoes (Figure 4). Several mosquito species primarily belonging to the Aedes genus are potential transmission vectors for ZIKV virus including Aedes africanus, Aedes luteocephalus, Aedes

furcifer, and Aedes vittatus, which are likely enzootic vectors in Africa 24. A recent study

indicates that up to 16% of some populations of African NHPs have been exposed to ZIKV, even in areas where ZIKV infections in humans have not been observed 25_{. Aedes aegypti and} Aedes albopictus are the major vectors of ZIKV transmission in nearly all known urban ZIKV

outbreaks, although two other Aedes species, Aedes hensilli and Aedes polynesiensis, were reported to be vectors in outbreaks in Pacific Islands 19,26_{. Aedes aegypti is widely distributed}

throughout the tropical and subtropical world. According to their distribution, Africa, Asia, Southern Europe, Americas and Oceania are susceptible areas for ZIKV dissemination 27_.

An urban transmission cycle was also hypothesized due to the 2007 outbreak in the Yap state despite absence of monkeys in this area and the scale of the recent outbreak in Brazil 22_{. Indeed, a possible sexual transmission of ZIKV was initially reported in 2008} 28_.

Thereafter other studies have shown that high concentrations of ZIKV ribonucleic acid (RNA) were detected in saliva, breast milk, urine and semen 29–31_{. Although the presence of ZIKV}

16 antigens has been detected within the head of spermatocytes 32 _{from ZIKV-infected man and}

ZIKV RNA has been detected in the head and flagella of spermatocytes from inoculated mouse

33_{, it is unclear which semen components contain infectious viruses. However studies have}

shown the presence of ZIKV RNA in the sperm of men without spermatocytes 34,35_{. Moreover,}

another study in mouse model revealed that vaginal tract could be a site for ZIKV replication

36_.

Moreover, ZIKV infection has been associated with a vertical transmission from ZIKV-infected mothers to fetuses with the ability of ZIKV to cross the human fetal-placental barrier

37_{. Although the precise mechanism with which ZIKV crosses the human fetal-placental barrier}

and the precise pathogenesis of congenital transmission remain poorly understood, sexual transmission and ascending vaginal infection leading to congenital ZIKV infection have been demonstrated in a mouse study 36_{. Furthermore, ZIKV RNA has been found in fetal and}

neonatal brain, amniotic fluid and in placenta of women who have acquired ZIKV infection during the first trimester of pregnancy, which could lead in some cases to abortion or fetal abnormalities (Figure 4) 36,38–40_.

Figure 4. Reported forms of ZIKV transmission.

The virus originates with nonhuman primates in tropical rainforests but can infect humans 23_.

c. Epidemiology

Until 2007, only some sporadic human disease cases were reported from countries in Africa and Southeast Asia 11,13,41_{. Then in 2007, the first documented ZIKV disease outbreak}

in the isolated islands of Yap was reported. In 2013, an outbreak began in French Polynesia, and the virus subsequently spread to other Pacific islands with more than 30 000 cases, some with neurologic complications 18_{. Cumulatively, as of March 2017, a total of 84 countries or}

territories globally had reported autochthonous mosquito-borne ZIKV transmission, including 61 countries or territories with new introduction of ZIKV since January 2015 42_{. Currently, ZIKV}

17

Figure 5. Areas of ZIKV introduction.

Countries and territories with confirmed Zika virus cases. Adapted from 42_.

Up to 1,5 million of ZIKV cases were reported in Brazil and an increase of Guillain-Barré syndrome and microcephaly have been observed in endemic areas. Several factors explain the worldwide emergence of ZIKV. Like other emergent viruses such as DENV and Chikungunya (CHIKV), genetic changes in the virus resulted in emergence of a virus strain with increased transmissibility leading to greater epidemic potential and likely virulence 43–47_.

Moreover, emergence and spread of ZIKV was facilitated by the global demographic, social and technological trends of population growth and urbanization with lack of effective mosquito control in urban areas, which provided optimal conditions leading to increased transmission and spread of the viruses and their mosquito vectors 48,49_{. Moreover, unlike other flaviviruses,}

ZIKV has the distinction of being transmitted sexually 28,50 _{and congenitally} 37_{, increasing its}

transmission possibilities. Indeed, an infected individual can in turn become a vector of transmission and infect healthy individuals. However, these modes of transmission remain minor compared to the transmission dependent on the Aedes vector.

d. Pathogenesis

ZIKV infection is reported to be symptomatic in 18% of cases only. For symptomatic cases, ZIKV infection usually presents itself as an influenza-like syndrome, often mistaken with other arboviral infections like dengue or chikungunya. Infected individuals present symptoms such as acute febrile illness with a low-grade fever between 37.8°C and 38.5°C, arthralgia, notably of small joints of hands and feet, with possible swollen joints, myalgia, headache, retroocular headaches, conjunctivitis, and cutaneous maculopapular rash. Digestive troubles

18 (abdominal pain, diarrhoea, constipation), mucous membrane ulcerations (aphthae), and pruritus can be more rarely observed 11,51,52_.

Since the ZIKV epidemic in French Polynesia in 2013, a more severe form of symptoms has been related among the infected individuals. Indeed, clinical symptoms are not restricted to flu-like illness but ZIKV infection has been associated with severe neurological complications such as meningoencephalitis, myelitis, and congenital microcephaly. These complications appear in the weeks or months following the acute febrile illness. In adults, ZIKV infection has been associated with Guillain-Barre syndrome (GBS), an immune-mediated disorder of the peripheral nervous system that manifests as acute onset of ascending paralysis and sensory symptoms 53_{. Retrospective studies reported an increase of GBS cases in ZIKV}

endemic areas. Indeed, from early 2013 to late 2014 in French Polynesia, on the registered 8750 suspected cases of ZIKV disease, 74 cases presented with neurological syndromes with 42 corresponding to GBS (higher GBS annual average of 3–8 cases) 54_{. Similarly, between}

2015 and 2016, clusters of GBS emerged shortly after the ZIKV outbreak in the Americas, in a pattern that highlighted a temporal and geographical association between GBS cases and ZIKV transmission. Overall, based on epidemiological studies, in 2016 the estimated GBS incidence increased from 2.0- to 9.8-fold in 7 countries in the Americas affected by the ZIKV epidemic 55_{. ZIKV infection has also been associated with an induction of microcephaly in the}

fetus of infected mothers. Moreover, even without cerebral abnormalities, ZIKV infection can also causes fetal growth problems and placental-related dysfunctions.

It is estimated that microcephaly and GBS occur in 1% and 0.02% of cases respectively 18,56_{. Neuronal disorders and microcephaly can be explained by ZIKV ability to}

preferentially infect neural progenitor cells 40,57_{. Microcephaly results from ZIKV replication in}

neural stem cells in the developing brain leading to cell cycle arrest and apoptosis 58_{. In addition}

to targeting progenitor cells in the developing brain, ZIKV can infect neural progenitors in the adult mouse brain, resulting in death and inhibition of their proliferation 59_.

e. Treatment and vaccines

There are currently no specific antiviral agents, vaccine, or prophylaxis for ZIKV.Due to its rapid spread, finding antiviral strategies against ZIKV has been an objective for the scientific community. Vaccine development efforts for ZIKV have been substantially informed by the prior development of efficacious vaccines against related flaviviruses including YFV, TBEV, JEV and DENV 60,61_{. There are more than 40 vaccine candidates in preclinical}

development whose seven vaccine candidates are being evaluated in clinical trials. The most rapid strategy to develop a vaccine is to use of deoxyribonucleic acid (DNA) or messenger

19 RNA (mRNA) encoding prM-E genes from recent or consensus ZIKV strains. Nevertheless, several groups have attempted to develop whole-inactivated virus vaccines, including one able to elicit robust neutralizing antibody responses and a protection against ZIKV infection in rhesus macaques and immunocompetent mice 62,63_{. Another strategy that has been attempted}

has involved vector-based that express ZIKV genes in the context of replication-competent or replication-defective viral vectors 62_{. These vaccines candidates are currently in development}

but have not yet been evaluated in humans 64_{. Furthermore, another strategy based on}

nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) has been engineered by two groups and could be a new and promising vaccine candidate against ZIKV 65,66_{. Indeed,}

these studies were demonstrated that a single intradermal immunization of these LNPs encoding the pre-membrane and envelope glycoproteins of ZIKV elicited potent and durable neutralizing antibody responses in mice and non-human primates 65 _{but also that by generating}

LNPs with a modified mRNA coding for ZIKV prM-E, whose fusion loop epitope conserved in protein E was destroyed, attributed protection to ZIKV in cells or mice while diminish the production of antibodies enhancing the infection with DENV 66_{. Therapeutic antibodies}

represent an attractive approach for ZIKV therapy. Studies showed that a human monoclonal antibody against viral E protein, to inhibit various strains from African and Asian-American lineages in cell culture exhibited efficacy in pregnant and nonpregnant mice 67_{. Moreover,}

another human monoclonal antibody, targeting viral E domain III, was reported to protect mice from lethal infection when given one day before or one day after infection 68_{. Nevertheless, an}

issue that needs to be addressed is the antibody-dependent enhancement (ADE). Indeed, studies revealed that the poorly neutralizing antibodies against DENV enhance ZIKV infection by ADE and vice versa 68–70_{. Furthermore, a drug strategy against ZIKV was explored. Indeed,}

two major categories of small molecule inhibitors have been reported for ZIKV. Studies showed a modest in vitro and in vivo efficacy with the nucleoside/nucleotide inhibitors 71–73_{. In addition,}

as nucleoside/nucleotide inhibitors possess a large broad antiviral spectrum against closely related viruses, they may have antiviral activity for other viruses of the same genus. Indeed, two of the four nucleoside inhibitors identified for ZIKV, present potent anti–DENV activities

20

2. Dengue Virus

a. History and emergence of Dengue Virus

The origins of the word dengue would derive from the Swahili phrase "Ka-dinga pepo", meaning a disease characterized by cramp-like seizure. The Swahili word "dinga" has its origin in the Spanish word "dengue" used to describe the gait of a person suffering the bone pain of dengue fever (DF). During the 1800s, the word was introduced by African slaves in the Caribbean, and progressively changed to “dengue” 76_{. Serological and phylogenetic studies}

have shown that this virus would not have an African origin, but an Asian one, and subsequently spread to the African continent. 77_{. Indeed, although dengue-associated}

symptoms are almost indistinguishable from those caused by other viral agents such as Chikungunya, first record of a case of probable DF can be found in a Chinese medical encyclopaedia from the Jin Dynasty (265–420 AD) which referred to a “water poison” associated with flying insects.

Then in the 1780s, first recognized dengue epidemics occurred almost simultaneously in Asia, Africa, and North America in the 1780s, shortly after the identification and naming of the disease in 1779. Based on a case in the 1780s, Benjamin Rush published the first confirmed case report on DF in 1789 and was coined the term "breakbone fever" because of the symptoms of myalgia and arthralgia. Initially, these cases were associated with flying insects developing in water reservoirs. It is only in the beginning of the twentieth century that

Aedes aegypti was identified as the main transmitting vector of the virus 76. Resurgence cases

of dengue among the troops deployed in endemic areas during World War II led to the isolation of the first two serotypes of DENV (DENV-1 and DENV-2) in the Pacific 78_{, then DENV-3 and}

DENV-4 in the 1950s in South-East Asia 79_.

b. Transmission

DENV is an arbovirus and is transmitted to the host by mosquito arthropods. There are two main transmission cycles described for DENV. The transmission of the virus in its simplest form involves the ingestion of viraemic blood by mosquitoes and the transition to a second sensitive human host is the urban cycle. Aedes aegypti mosquitoes have been identified as the main vectors of DENV in this cycle. Originally, Aedes aegypti was found only in Africa. It is now a mosquito adapted to urban areas that has spread widely in the tropical and subtropical world areas 80_{. However, two Asian species of Aedes, Aedes albopictus and}

21

Aedes polynesiensis, have also been identified to contribute to the transmission of DENV with

less efficacy and have been characterized as secondary vectors 81_{. Aedes aegypti has been}

shown to be adapted to humans, preferring to feed on them and to lay their eggs in artificial water storage containers that surround human habitats.

After ingestion of blood from an infected human during the viraemic phase of the illness (2 days before and 4-5 days after the onset of fever), by the female mosquito, an incubation period of 8-10 days is required for viral replication and internal dissemination in the mosquito before virus appears in the saliva and transmission on refeeding can occur. As the blood meal stimulates oviposition by the female mosquito, which undergoes at least one, and often more, reproductive cycles during the extrinsic incubation period, there is an opportunity for virus to enter the egg and be passed to the next generation of mosquitoes 82_{. Furthermore,}

female mosquito remains infected for life and can feed on several individuals in a short period of time, which contribute to the fast spread of DENV (Figure 6) 83_.

In tropical Asia and West Africa, DENV is also transmitted between non-human primates and arboreal Aedes spp. Mosquitoes, which is the sylvatic cycle. DENV comes from sylvatic cycles between Aedes spp. mosquitoes and especially the African green monkey (Chlorocebus sabaeus) and the Guinea baboon (Papio papio). As in the urban cycle, transovarian transmission is possible 84_{. Occasionally, when humans have been exposed to}

sylvatic DENV, hematophagous Aedes mosquitoes may transmit DENV to rural human communities, but these are considered accidental contacts 83_{. In addition, phylogenetic}

analysis identified four chains of transmission showing that each of the four DENV lines jumped from a non-human primate reservoir to a human, which explains the four serotypes represented 84_.

22

Figure 6. Transmission cycles of DENV.

DENV transmission is sustained between non-human primates and several species of Aedes mosquitoes in the salvatic cycle. Occasionally, when a human is exposed to this cycle, he may be infected during the mosquito's blood meal. DENV transmission will then be sustained between humans and essentially the vector Aedes aegypti in the urban cycle. TOT: transovarial transmission. Adapted from 84_.

c. Epidemiology

Among arboviroses, DF is distributed worldwide and fastly progresses. Until the 1970s, only nine countries were reported with sporadic outbreaks of dengue fever responsible for less than 200,000 cases per year. Furthermore, concomitant circulation of more than one serotype of DENV was restricted to Central America, South East Asia and Western Africa. Since then, the number of affected countries has grown steadily with a doubling of cases every decade until 2000 and then almost doubling every 5 years. Currently, the number of estimated annual cases is 50 million. Each year, DENV is responsible for more than 500,000 DF or dengue haemorrhagic fever (DHF) and 20 to 25,000 deaths, mainly in children. Dengue shock syndromes (DSS) were reported in almost 60 countries 85_{. In recent years, there has been an}

intensification of dengue epidemics in urban areas and an extension to rural areas. All continents are currently affected and more particularly South America, Asia and Pacific islands where 2.5 billion individuals are at risk of infection (Figure 7). Furthermore, the number of DENV infections per year worldwide has been estimated to 390 million, of which 96 million led to symptomatic dengue cases 86_.

23

Figure 7. Global Dengue Risk.

Areas at risk concentrate in the intertropical zone in 2014 according to best estimates from 86_{. Red: areas at high}

risk of infection; Pink: areas at medium risk of infection; Light pink: areas at low risk of infection. Adapted from 87_.

d. Pathogenesis

In the majority of cases, an infection with any of the DENV serotype remains asymptomatic (~ 80% of cases). However, in some cases, an infection by this virus can result in a wide spectrum of clinical symptoms, ranging from a mild flu-like syndrome, termed as DF, to the most severe forms of the disease which are characterized by coagulopathy, increased vascular fragility, and permeability, termed as DHF. This form of the disease may progress to hypovolemic shock, termed as DSS 88_.

In older children, adolescents and adults, DF is characterized by the rapid onset of fever in combination with severe headache, retro-orbital pain, myalgia, athralgia, gastrointestinal discomfort, and usually rash. Minor hemorrhagic manifestations may occur in the form of petechiae, epistaxis, and gingival bleeding. Moreover, a leukopenia is commonly observed whereas a thrombocytopenia may occasionally be observed in DF. This forms of the disease typically lasts for 7-14 days 89,90_.

DHF is classified in four grades. DHF grades I and II represent relatively mild cases without shock, whereas grade III and IV cases are more severe and accompanied by shock. DHF is characterized by all symptoms in DF, in combination with haemorrhagic manifestations, thrombocytopenia and vascular and plasma leakage inducing an increasing haemoconcentration or fluid effusion in chest or abdominal cavities 4_{. DHF could progress in}

DSS if the leakage and/or the bleeding leads to a critical volume of plasma lost that could be sufficient to induce shock, which is characterized by a rapid, weak pulse or hypotension with cold, clammy skin in the early stage, corresponding to grade III. A stage of profound shock

24 may set in, corresponding to grade IV, in which pulse and blood pressure become undetectable, resulting in death within 12 to 36 hours after onset of shock.

The pathogenesis of DENV infection is not well understood. Many hypotheses can explain the increase in disease severity. Some factors seem to influence the severity of the disease. Cell and tissue tropism of DENV may have a major impact on the severity of disease. The immune system, the liver, and endothelial cell linings of blood vessels are three organ systems that could be playing an important role in the pathogenesis. Moreover, certain DENV strains are responsible for more severe disease. In 1981, there was a correlation between an outbreak of DHF in the Americas and the Southeast Asian genotype of DENV-2 introduction

91_{. Then, activation of the complement system appears to increase the severity of the disease.}

Indeed, studies have reported that in patients with DSS, high levels of the activation products C3a and C5a are measured in the plasma, followed by an accelerated consumption and a marked reduction of the complement components 92,93_{. An autoimmune role also seems to play}

a role 94–96_{. Indeed, in DHF/DSS there is an increase of markers of immune activation and}

complement activation, pointing out an autoimmune process 97_{. Furthermore, the studies of}

polymorphism of candidate genes have outlined the association between host genetic variations and dengue severity 98–100_{. Indeed, the frequency of DDX58 rs669260 allele was}

significantly higher in DHF cases than in DF cases 101_{. Moreover, the OSBPL10 expression is}

significantly lower in Africans than Europeans. The DENV infection in OSBPL10 knockdown cells led to a significant reduction in DENV replication, which supports that the African-ancestry-conferred resistance to DHF, in comparison with European background 99_.

Furthermore, primary infection by one of the four DENV serotype allows the generation of type specific and serotype cross-reactive antibodies that confer a durable immunity against re-infection by a homologous DENV serotype. Nevertheless, a secondary infection may constitute the most important risk factor for severe disease 102_{. Moreover, the}

infants born to dengue-immune mothers can develop an infant DHF in case of primary infection, which consists of the severe dengue disease. These observations were attributed to a phenomenon referred as ADE.

ADE consists of pre-existing neutralizing antibodies against a DENV serotype that promote entry of another DENV serotype into myeloid cells, such as monocytes, macrophages and dendritic cells (DC) which express Fcγ receptors (FcγR) 102,103_{. This mechanism allows an}

increase in viral burden 104,105 _{and promotes a “cytokine storm” and vascular leakage, which}

requires interactions of the Fc region of antibody with FcγR 106_{. Apart from ADE, the ligation of}

FcγR on myeloid or mast cells by DENV immune complexes may modulate host immunity and disease pathogenesis by increasing interleukin-10 (IL-10) production, skewing CD4+ T cell responses, or promoting degranulation of vasoactive molecules that enhance capillary leakage

25

107,108_{. However, the mechanism is not well understood and remains the subject of numerous}

studies.

e. Treatment and vaccines

Numerous strategies to create DENV vaccines have been explored. However, given that ADE increase the severity of the dengue disease, the engineered vaccine must be tetravalent and induce the stimulation of immune response to the four serotypes. Three live-attenuated tetravalent DENV vaccine candidates were being evaluated in large clinical trials. The CYD tetravalent, live-attenuated dengue vaccine candidate (CYD-TDV-Dengvaxia®), which contains four chimeric viruses in which the structural genes of the YFV vaccine virus (YFP-17D) were replaced with those of each DENV serotype, was the most promising. Indeed, this candidate showed a protective effect varying from 35% at 78% according to DENV serotype 109_{, with the best immunogenicity for DENV-4 and the worst for DENV-2. However, a}

large scale clinical efficacy trial of this candidate has allowed to show that this vaccine reduces the overall risk of severe dengue and hospitalizations in those who had a prior history of dengue infection, but for individuals who had never been infected before, the risk to develop a more severe form of the disease and hospitalizations was higher following vaccination with Dengvaxia® 110,111_{. Two additional vaccine candidates have advanced to phase II trials. First,}

the recombinant live-attenuated tetravalent dengue vaccine (TDV) candidate DENVax, is composed of an infectious clone-derived attenuated DENV-2 strain and three chimeric viruses that incorporate the structural genes of the other three serotypes 112_{. This candiate induces a}

tetravalent neutralizing antibody response in 44%–80% of recipients depending on the route of immunization and dose 112_{. As to the second vaccine candidate, composed of a mixture of}

modified full-length and chimeric DENV strains (TV005), studies were reported that a single dose was sufficient to elicit a neutralizing antibody response against all four DENV serotypes in 90% of recipients 113_{. Furthermore, live-attenuated, inactived viruses, virus like particles,}

recombinant proteins and DNA (prM and E genes) vaccines candidates are under development and clinical trials 87,114_.

Beyond vaccine development, other strategies have been pursued to control DENV infections, in particular to identify inhibitors of specific stages of the life cycle of DENV 115_.

Efforts have focused on finding antiviral drugs that target viral enzymes, the protease and helicase proteins NS3 and the RNA-dependent RNA polymerase and methyltransferase NS5. Moreover, efforts have also focused to identify antiviral drugs against the integral membrane proteins NS2A and NS4B required for replication as well as inhibitors of the fusogenic viral E protein 116,117_{. However, because against viral proteins could promote resistant variants}

26 expansion, identifying inhibitors that targeting host molecules required for DENV infectivity has been proposed as alternative strategy. Moreover, another considered strategy consisting in reducing DENV transmission by limiting infection in the mosquito host 118–121_.

III. Flavivirus genome and viral particle organization

1. Genome organization

Flavivirus genome is a single-stranded positive sense RNA of approximately 11 kilobases in length. This viral RNA contains a unique open reading frame (ORF) flanked by two untranslated regions (UTRs) (Figure 8). During infection, the viral genome serves as mRNA for translation, and subsequently, as template for RNA replication. The newly synthesized RNA was used for new rounds of translation or as substrate for coating. Efficient utilization of the genome during these processes was temporally regulated to ensure viral spread. This regulation is mediated by RNA elements present in the coding and non-coding regions of the viral genome acting as promoters, enhancers and repressors of the viral processes. In addition, during infection the viral RNA participates in triggering or avoiding the antiviral host response.

Translation of the single ORF of the viral genome at the rough endoplasmic reticulum (ER) membrane generates a large polyprotein that is processed co- and post-translationally cleaved by cellular and viral proteases. This generates three structural proteins capsid (C), premembrane (prM) and envelope (E), which constitute the viral particle and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) involved in viral RNA replication, assembly and modulation of host responses (Figure 8).

27

Figure 8. Flavivirus genome organization and polyprotein processing.

RNA genome of flaviviruses is flanked by two UTRs and contains a single ORF. Translation of the single ORF at the rough endoplasmic reticulum (ER) membrane produces a large polyprotein that is cleaved co- and post-translationally into the mature proteins by viral and cellular proteases. The N-terminal of the polyprotein encodes the three structural proteins (C-prM-E), followed by seven non-structural (NS) proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). Adapted from 122_.

The genomic viral RNA has a type I 7-methylguanosine cap at its 5 'end (m7GpppAmp) which is followed by the conserved dinucleotide sequence AG. This structure allows it to mime the one of cellular mRNAs to initiate the translation of the viral genome 123_.

Type I 7-methylguanosine cap allows the binding of cellular factors on genomic viral RNA to form a scaffold complex, allowing the recruitment of the small ribosomal subunit and the initiation of protein synthesis 124,125_{. The 5’ UTR (95 to 132 bases in length) contains two RNA}

domains with distinct functions during viral RNA synthesis. The first domain of ~70 nucleotides forms a structure folding into a large stem-loop (SLA). This structure acts as the promoter for the viral RNA-dependant RNA polymerase NS5 (RdRp), which directly bind with SLA and is necessary for viral RNA synthesis 126_{. The second domain forms a structure folding into a short}

stem loop (SLB), which contains essential sequences for long-range RNA-RNA interaction and genome replication. The two domains are separated by an oligo(U) sequence, which functions as spacer for proper function of the two stem loops. These regions are highly conserved among flaviviruses despite the lack of sequence homology between them 127_.

Flavivirus RNA lacks a poly(A) tail at its 3' end and terminates with the conserved dinucleotide CUOH 128. The 3’UTR is variable among flaviviruses (340 to 700 nucleotides)

contains specific structures that also play crucial roles in viral RNA synthesis. This region is divided in three domains: domain I which is located immediately after the stop codon and is

28 the most variable region within the viral 3'UTR (VR), domain II that includes a characteristic dumbbell (DB) structure which is duplicated in tandem and contain conserved sequences named cyclization sequence 2 (CS2) and repeat CS2 (RCS2), and domain III that is the most conserved region of the 3'UTR, bearing a CS1 element followed by a terminal stem-loop structure (3'SL) that is necessary for genome replication 129_{. CS1 contains a sequence involved}

in long range RNA-RNA interaction between the ends of the viral genome 130_{. The 3' terminal}

structure contains a short stem loop of 14 nucleotides (sHP) followed by a large stem loop of 79 nucleotides.

The linear RNA form serves as mRNA for polyprotein precursor translation. However, the CS sequences present in the 5’UTR and 3’UTR allow the circularization of the viral genome through long-range 5’-3’ RNA-RNA interactions that are required for minus strand synthesis (Figure 9A) 131_{. Indeed, during RNA viral replication, viral NS5 protein interacts with the SLA}

of 5’UTR via its methyltransferase region bringing the RdRp region of this protein close to the 3' end of the genome, which allows the viral minus strand synthesis 123_{. Moreover, it has been}

hypothesized that high concentrations of viral RNA genome in infected cells, could form concatemers and non-covalent interactions between separate viral genomes, no longer requiring to switch between linear and circularized forms, and by 3'-5 pairing could promote an increase in the minus strand synthesis and the efficiency of replication (Figure 9B and 9C) 132_.

29

Figure 9. Flavivirus genome 5'-3' long distance interaction.

Presentation of different DENV (+)-sense RNA conformational forms. (A) The linear DENV RNA genome is circularized through conserved regions present in 5’ and 3’ UTRs through the interaction of the 8 nucleotides cyclization sequence (CS, in purple) in 3’ with its exact complement sequence near the 5’ end, located in the capsid coding region. 5’ Upstream AUG Region (UAR, in orange) and 5’ Downstream AUG Region (DAR, in green) interact with complementary 3’ sequences. (B) Two DENV genome can form an antiparallel homodimer molecule by intermolecular base pairing using the same complementary regions. (C) A head-to-tail concatemer is also possible through intermolecular base pairing. Adapted from 132_.

2. Non-structural (NS) proteins

a. NS1

In 1970, experiments to determine the physical and biological properties of DENV and associated antigens described NS1 for the first time as a soluble complement-fixing antigen

133,134_{. NS1 is involved in immune evasion and pathogenesis by interacting with components}

from both the innate and adaptive immune systems. Moreover, high circulating levels NS1 correlate with the dengue disease severity 135_.

NS1 is cleaved by an unknown protease in ER lumen and N-linked glycosylated in two or three sites, depending on the virus. Upon NS1 release from the E and NS2A by