Jean-Pierre Levraud
email: jean-pierre.levraud@pasteur.fr

Pierre Boudinot
email: pierre.boudinot@jouy.inra.fr

J.-P Levraud

Eric Freed

Michael Gale

Keywords:

Innate immunity constitutes the first line of the host defense after pathogen invasion. Viruses trigger the expression of interferons (IFNs). These master antiviral cytokines induce in turn a large number of interferon-stimulated genes, which possess diverse effector and regulatory functions. The IFN system is conserved in all tetrapods as well as in fishes, but not in tunicates or in the lancelet, suggesting that it originated in early vertebrates. Viral diseases are an important concern of fish aquaculture, which is why fish viruses and antiviral responses have been studied mostly in species of commercial value, such as salmonids. More recently, there has been an interest in the use of more tractable model fish species, notably the zebrafish. Progress in genomics now makes it possible to get a relatively complete image of the genes involved in innate antiviral responses in fish. In this review, by comparing the IFN system between teleosts and mammals, we will focus on its evolution in vertebrates.

ACKNOWLEDGMENTS

Of all the Chapters included in this Thesis, I must admit that this certainly represents the one I had more difficulties writing. That is because I feel that a simple thank you to all the amazing people I encountered during my three years of PhD feels like very little as compared to what I gained from each of them. I hope therefore that everyone will excuse me if I don't manage to properly convey how grateful I am for all your help. I should definitely start by thanking my two amazing supervisors, Jean-Pierre Levraud and Pierre Boudinot. I should thank you not only because of the opportunity you gave me when you accepted my candidature but also because of all the help and support you have provided on a daily basis for over three years now. You have shown me over and over again that you care not only as supervisors but also as friends. You are both extremely talented, hard-working and an example to follow. I wish you both all the best for your future projects as well as personal achievements.

Christelle and Nuno, my two great friends. I don't remember how many laughs we have shared and certainly no words can express how grateful I am for the time I got to spend with you. From your help with my project, to your suggestions and advice on a more personal level, you made my experience in Paris an amazing one. I hope you will be able to reach all your future goals and that you won't lose your positive attitude and amazing personalities.

I want to thank all the awesome people in the two labs: Aurelie, Brigitte, Elina, Armel, Dominique, Jean-Francois, Luc, Eric, Tatiana, and Abdenour from INRA and Valerie, Emma, Jose, Laurent, Catherine, Emi, Mylene, Mireille, Pedro, Anne, Philippe, Laurence, Maxence, and Ramil. Thank you for the free French lessons! Thank you for caring for me anytime I was feeling down! Thank you for VIII listening and giving me your advice! And thank you for being funny and easy to work with! You made for an entertaining working environment! Thanks to all the group of FishForPharma! It was amazing how we all got along so well from the very first meeting in Paris. From the supervisors, to the PhD students and Post-Docs, you all contributed in some way, either on a scientific or on a more personal level. Thank you all and wish you all the best! Thank you Leandro and Mathilde, for all the lunches and coffee breaks! I wish you all the best in life! Thank you Lavinia, for being the awesome person you are! Don't ever change! And a super good luck with you PhD! PS: Start writing the introduction now!  Thank you Anna, for the endless study sessions during University and the laughs and the great time together! I know you will achieve everything you set your mind to, therefore I'm not worried to know you'll soon be leaving for a far and likely very cold country! You are surrounded by amazing people, and that's because you yourself are amazing! Paolo! Thank you for keeping me sane during the last year and for being an amazing best friend for over 17! I wouldn't have gotten this far without you and I hope you will continue to be part of my life for the foreseeable future! And thanks to my family, for all the support and love you share on a daily basis!

RESUMÉ ÉTENDU DU PROJET DE THÈSE

De nombreux pathogènes sont neurotropes, posant un problème aigu de santé publique; on estime ainsi que près de la moitié des virus émergents peuvent causer des encéphalites ou d'autres atteintes neurologiques sévères. La compréhension des interactions qui sous-tendent le neurotropisme de ces pathogènes est d'une importance considérable, afin de guider les stratégies thérapeutiques et préventives pour limiter les conséquences de ces infections.

Le genre Alphavirus compte plusieurs virus neuro-invasifs et inclut par exemple le virus de l'encéphalite équine occidentale (WEEV), causant une mortalité d'environ 10%, et le virus du Chikungunya (CHIKV), qui s'est récemment propagé aux Caraïbes et en Polynésie, et qui cause de graves encéphalites chez les nouveaux-nés.

Le laboratoire où j'ai effectué une partie de ma thèse a décrit, par imagerie de l'organisme entier, la dissémination du CHIKV dans le modèle danio zébré (Danio rerio), pour mieux caractériser sa pathogènese ainsi que les reponses immunitaires activés chez le danio. Le danio zébré est un modèle bien établi pour étudier la biologie du développement des vertébrés. Ses larves petites, transparentes et faciles à anesthésier, sont favorables à des approches de microscopie non invasive, qui permettent de réaliser des observations à l'échelle d'un individu entier à des niveaux de résolution cellulaire et subcellulaire. De surcroît, les outils génétiques disponibles chez cette espèce ont permis le développement de nombreuses lignées transgéniques exprimant des protéines fluorescentes spécifiquement dans certaines cellules (e.g. neurones, cellules immunitaires). De telles lignées constituent des outils de choix pour évaluer la dynamique de la réponse immunitaire et la mort cellulaire neuronale au cours de l'infection virale. Ces atouts font du danio zébré un excellent modèle pour étudier les infections virales in vivo.

Au cours de mon projet, j'ai etudié l'entrée et la colonisation du système nerveux central (SNC) par un Alphavirus de la même famille de CHIKV, le virus Sindbis (SINV) dans le modèle danio zébré. SINV est transmis chez l'homme par piqûre X de moustique et provoque une maladie avec des symptômes tels que fièvre, éruption cutanée, douleurs articulaires et musculaires. De plus, le virus induit une forte réponse de l'hôte, sous forme de production d'interféron de type I. Malgré toutes les études conduites jusqu'à présent, ni le mécanisme d'entrée ni la réponse induite par l'infection dans le cerveau n'ont pu être visualisés en temps réel à l'échelle d'un organisme entier. Mon projet présentait donc plusieurs axes: 1) développer un modèle d'infection du virus Sindbis chez le danio zébré, 2) caractériser l'invasion du SNC par le virus par des techniques d'imagerie à haute résolution, 3) définir la voie d'entrée du virus dans le SNC, 4) évaluer la dynamique de la réponse immunitaire innée par l'étude de la réponse IFN et,enfin,[START_REF] Robertsen | Atlantic salmon interferon genes: cloning, sequence analysis, expression, and biological activity[END_REF] établir les mécanismes de persistance du virus dans les cellules neuronales.

Les études du virus SINV réalisées dans les modèles murins reposent essentiellement sur des injections par voie intracrâniale (IC), ce qui ne permet pas d'identifier la voie d'entrée dans le cerveau. Par conséquent, j'ai réalisé des injections par voie intraveineuse (IV) afin de pouvoir observer le passage de la périphérie au cerveau. Le suivi de la propagation du virus a été rendu possible par l'utilisation d'un ARN viral recombinant dans lequel est intégré la séquence de la protéine fluorescente verte 'GFP' exprimée dans les cellules cibles de l'infection lors de la réplication virale. L'utilisation de cette construction m'a permis de caractériser la progression de SINV chez le danio zébré et d'identifier les organes/tissus cibles que sont le vitellus, le foie, le coeur et enfin, le cerveau. De plus, j'ai pu montrer par des techniques de biologie moléculaire que SINV provoque une réponse interféron très forte dans les larves infectées, tout comme dans les modèles d'infection chez les mammifères. L'importance de cette réponse a été corroborée par la mortalité plus élevée que l'on observe dans les larves où la réponse interféron a été inactivée.

Les données rassemblées jusqu'à présent m'ont aussi permis d'identifier le mécanisme par lequel SINV se propage vers le cerveau. J'ai pu constater que SINV ne détruit ni n'infecte la barrière hémato-encéphalique, après infection d'un lignée transgénique permettant l'étude du système vasculaire; j'ai aussi montré qu'il n'exploite pas l'entrée naturelle dans le cerveau des leucocytes circulant comme les macrophages (tactique du 'Cheval de Troie'), puisque la progression XI de l'infection est inchangée lors de la déplétion sélective des macrophages. La primo-infection des neurones périphériques notamment au niveau des ganglions trigéminés semble indiquer que le virus se propage par un transport axonal du system nerveux périphérique vers le SNC.

Dans le cadre de la réponse immunitaire au niveau cellulaire, j'ai pu observer le rôle joué par les leucocytes, en particulier les neutrophiles, comme cellules productrices d'interféron: l'infection du cerveau, à partir d'une certaine taille du foyer, détermine le recrutement de ce type cellulaire. Cette réponse dans le système nerveux est particulièrement intéressante, si l'on considère que, chez le danio zébré tout comme chez les mammifères, les neutrophiles sont normalement exclus du cerveau.

Enfin, le danio zébré est devenu un modèle de choix courant pour réaliser des criblages pharmacologiques, et ces travaux permettent d'identifier de nouvelles molécules antibactériennes, antivirales ou anti-inflammatoires. Mon modèle est donc maintenant exploité pour évaluer les propriétés antivirales d'une drogue spécifique sélectionnée par des approches in vitro, en collaboration avec l'Unité « Populations Virales et Pathogenèse » à l'Institut Pasteur.

Compte tenu de la conservation entre les types cellulaires et les gènes de l'homme et du danio zébré, j'espère que ce travaille de thèse permettra d'identifier certains mécanismes généraux impliqués dans le neurotropisme, ce qui pourrait à plus long terme servir a la mise en place d'un prévention des encéphalites virales.

SUMMARY

The zebrafish (Danio rerio) is an important model organism, particularly for studies of development, cancer, neuroscience and more recently host-pathogen interactions. As opposed to other vertebrate model organisms, its optical clarity and ease of genetic manipulations allow to visualize highly dynamic cellular processes in vivo at the whole-organism scale. These assets make the zebrafish a perfect model for the study of viral infections in vivo, such as those caused by neurotropic viruses.

Viral infections that involve the Central Nervous System (CNS) are particularly serious, especially due to the limited number of effective treatments or prophylactic drugs. Moreover, the CNS is a particularly vulnerable region.

Encephalitis and meningitis are among the worst complications that can arise from a viral infection. This can be due to strong inflammatory responses, which are especially deleterious in the CNS, and must therefore be tightly and specifically regulated.

The aim of this project has been to gain insights in some of the interactions that determine encephalitis, by combining the optical properties offered by the zebrafish model with the neurotropic features of the widely-known Sindbis virus (SINV).

SINV is a single-stranded positive-polarity RNA virus belonging to the Togaviridae family, genus Alphavirus. Together with other medically important viruses (such as Dengue and Nipah virus), it is an arbovirus, thus transmitted to humans by arthropods -mostly represented by mosquito vectors. SINV has been extensively used in the laboratory, especially in murine models, with some highly encephalitogenic strains being developed for that host. Despite extensive studies in this model, the exact mechanism of entry in the CNS has never been fully elucidated. This is due mainly to difficulties in assessing the distribution of the virus and following the progression of the infection in real time in live animals.

This Thesis project has consisted therefore in: 1) the development of a SINV infection model in zebrafish larvae, 2) the characterization of SINV neuroinvasion XIV upon its inoculation in the bloodstream, thanks to the use of high-resolution microscopy, 3) the study of SINV mechanism of entry in the CNS, 4) the characterization of the innate immune response, both at the whole organism and organ-specific level, and 5) the characterization of SINV persistence mechanism in infected neuronal cells.

Thanks to the use of a SINV recombinant strain, engineered to express the green fluorescent protein "GFP" in infected cells upon viral replication, we have been able to follow the onset and the progression of the infection. We have identified several organs targeted by the virus, including the yolk, the heart, the liver, and eventually, the brain. Moreover, we have investigated the mechanism through which SINV reaches and infects the CNS. We have excluded propagation from the blood-brain barrier as well as infection of circulating leucocytes ("Trojan horse" mechanism) and have suggested infection of peripheral neurons and subsequent axonal transport to the CNS as SINV entry mechanism. These results have been reported in the submitted article. Furthermore, the activation of the host response at early times post-infection as well as CNS-specific responses have been investigated. In particular, at the cellular level, we have identified neutrophils as the main IFN-producing cells and are currently investigating their recruitment to the brain, upon extensive CNS infection in zebrafish larvae.

In addition to these in vivo analyses of infection, several tools have been developed for the project: production of various recombinant SINV viruses (leading to interesting findings of strain-dependent neuroinvasiveness), and in vitro culture of primary zebrafish neurons and glial cells.

Finally, we initiated a collaboration with the "Viral population and Pathogenesis" Unit at Pasteur Institute, to evaluate the effect of antiviral compounds on SINV-infected larvae.

Thanks to the conservation between zebrafish and human genes and cell types, we hope this project will shed more light on the mechanisms of viral neurotropism. This, in turn, will be helpful long-term to set up strategies for the prevention of viral-induced encephalitis. "Riper matter broke forth towards the ear. Subsequently, there were sweats (lesions) on the head for a long time."

LIST OF TABLES AND FIGURES

Hippocrates Volume VII, Epidemics II, Harvard University Press, 1994 In this passage extracted from Epidemics II, the Greek physician Hippocrates describes a disease of unknown origin affecting one of his patients and causing lesions spreading on the head. It is the beginning of the 4 th century BC when

Hippocrates first reports a case of a herpes-induced brain infection.

Despite being well known since ancient times, infectious diseases of the Central Nervous System (CNS) did not cease to pose a major health threat to the human population, as exemplified by the number of pathogens that can affect the CNS.

Table 1 provides a general overview of neurotropic viruses, based on their classification and properties (Table I1.1).

With the exception of a few specialized human viruses (e.g. Poliovirus and herpesviruses), persistent or "stable" neuroinvasion is generally rare. On the other hand, acute infections of the CNS are more often caused by zoonotic diseases, for which humans normally represent a "dead-end host" (Gubler, 2001).

It has recently been estimated that nearly 50% of emerging viruses are characterized by serious neurological clinical manifestations [START_REF] Olival | The ecology of emerging neurotropic viruses[END_REF]. Contacts between humans and animals, especially wild ones, are an important factor for the onset and transmission of viral Emerging Infectious Diseases (EIDs), 75% of which are of zoonotic origin [START_REF] Taylor | Risk factors for human disease emergence[END_REF]. It has been shown, for example, that bushmeat hunters in Africa are at high risk of contracting novel viral zoonoses [START_REF] Wolfe | Naturally acquired Simian retrovirus infections in central African hunters[END_REF].

Table I1.1. Classification and CNS disease of neurotropic viruses. dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; ssRNA-RT, RNA reverse transcribing. In bold, neurotropic arboviruses. Adapted from [START_REF] Koyuncu | Virus infections in the nervous system[END_REF].

Changes in ecological, environmental, and demographic factors also contribute to the spreading of these diseases. Moreover, of all viral EIDs 40% are arboviruses (Arthropod-Borne Viruses) -pathogens transmitted by arthropods. Increased exposure to mosquitoes and other arthropod vectors, adjustment of a vector to new ecological niches [START_REF] Roche | The spread of Aedes albopictus in metropolitan France: Contribution of environmental drivers and human activities and predictions for a near future[END_REF] or adaptation of a pathogen to new vectors greatly increase viral EIDs expansion. The adaptation of Chikungunya virus (CHIKV) to the mosquito Aedes albopictus since 2004, for example, greatly contributed to its re-emergence in various parts of the world [START_REF] Tsetsarkin | Sequential adaptive mutations enhance efficient vector switching by Chikungunya virus and its epidemic emergence[END_REF].

Reducing vector prevalence and vector avoidance are certainly the first line of defense against arboviral nervous system infections. However, new outbreaks of both new and old pathogens are almost certain to occur in the future, and novel treatments -possibly with broad relevance to many viruses -should also be part of our global health strategy. To do so, a thorough understanding of the mechanisms of disease is necessary and animal models play a key role in deciphering virus-induced pathogenesis.

The following sections include a brief overview of neurotropic arboviruses, the neurological pathogenesis they can induce, the several mechanisms of entry neurotropic viruses can employ to reach and spread in the CNS, and the defense mechanisms that are readily activated in the CNS upon infection.

Neurotropic Arboviruses

Arboviruses that can reach and affect the nervous system mostly include Orthomyxoviridae (Influenza virus), or to the Reoviridae (Colorado Tick Fever virus) families (Table I1.1).

Neurotropic viruses of the Togaviridae family include Alphaviruses such as CHIKV, Sindbis virus (SINV), Eastern Equine Encephalitis virus (EEEV),

Western Equine Encephalitis virus (WEEV), and Venezuelan Equine Encephalitis virus (VEEV). Their structure, life cycle, and pathology are further described in Chapter II.

Arboviruses are found worldwide and can be transmitted to several vertebrate hosts by blood-feeding arthropods, including mosquitoes, biting flies, ticks, mites and lice. They are normally maintained in complex life cycles involving vertebrate hosts and arthropod vectors (Figure I1.1). Epidemics in humans and domestic animals occur when the virus comes in close contact with a bridge vector. Nevertheless, humans are generally dead-end hosts.

They do not in fact contribute to the distribution/spreading of the disease, since they do not normally develop sufficient levels of viremia to infect an arthropod, such as in the case of EEEV (Figure I1.2).

Figure I1.2. Transmission cycle of Eastern Equine Encephalitis virus.

The enzootic cyle occurs in hardwood swamps where the vector transmits the virus among passerine birds. Other vectors are believed to transmit the virus to humans and horses. These develop little viremia and do not contribute to amplification. Adapted from [START_REF] Weaver | Host range, amplification and arboviral disease emergence[END_REF].

Viruses such as DENV or CHIKV represent important exceptions to this rule.

They can reach high levels of viremia and can be transmitted from humans to other humans by mosquitoes (urban cycle) [START_REF] Weaver | Transmission cycles, host range, evolution and emergence of arboviral disease[END_REF].

The incidence of arboviral-induced neuroinvasion varies annually depending on vector abundance, geographical location, climatic conditions, and human behavior. As disclosed by the Centers for Disease Control and Prevention (CDC), in 2013 over 2600 cases of arboviral diseases were notified in the United States, 53% of which were reported as neuroinvasive. WNV was the most prevalent neuroinvasive arbovirus, with over 2400 reported cases (Lindsey et al., 2014).

Preliminary data on WNV infection in 2015 are in accordance with previous reports, showing over 60% neuroinvasiveness (Figure I1.3).

Arbovirus-Induced Neurological Pathogenesis

As can be expected, morbidity and mortality from CNS arboviral infections are significantly high and, depending on the pathogen, can lead to fatality rates ranging between 1% and 70% [START_REF] Hollidge | Arboviral encephalitides: transmission, emergence, and pathogenesis[END_REF]. When not fatal, these infections lead to long-term sequelae in 90% of affected patients and include conditions such as cognitive deficits, blindness, epilepsy, flaccid paralysis of limbs, focal neurologic deficits, and permanent vegetative state [START_REF] Rust | Human arboviral encephalitis[END_REF].

The neurological manifestations that can arise upon infection of vector-borne viruses include myelitis, encephalomyelitis, neuritis, myositis, and more often meningitis or encephalitis. WNV, for example, most commonly induces encephalitis but can also lead, in some cases, to meningitis and/or acute flaccid paralysis [START_REF] Tyler | Emerging viral infections of the central nervous system: Part 1[END_REF].

While meningitis involves inflammation of the tissues lining the brain, encephalitis is an acute inflammation of the brain parenchyma, and myelitis an inflammation of the spinal cord, both typically due to the infection of neurons belonging to the CNS. This in turn leads to neuronal cell death. Human

Enteroviruses and Alphaviruses are known to induce apoptosis in infected neurons [START_REF] Kuo | Infection with Enterovirus 71 or expression of its 2A protease induces apoptotic cell death[END_REF][START_REF] Jan | Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication[END_REF]. In the case of Flaviviruses, the induced cellular immune response upon CNS infection is instead considered to play a greater role in disease pathogenesis, such is the case of Japanese Encephalitis virus (JEV) [START_REF] German | A preliminary neuropathological study of Japanese encephalitis in humans and a mouse model[END_REF]. Other neurotropic viruses (not restricted to arboviruses) can cause damage by disrupting the blood-brain barrier (BBB), inducing autoimmune attack on specific cell types, altering gene expression, causing cell fusion, altering neuronal migration, reducing neural progenitor replication, and blocking cerebrospinal fluid (CSF) generation.

Despite many years of study, it is still not completely understood why only a certain percentage of patients develop serious neurological symptoms after infection. A fundamental factor certainly involves the efficiency of one's immune system in eliminating the pathogen from the whole body. It has been shown, for example, that mutations in the ccr5 gene (a chemokine receptor and co-receptor for Human Immunodeficiency virus (HIV)) increase the incidence of symptomatic WNV infection (Glass et al., 2006). Another important aspect to consider is the age of the patient at the time of infection. Intrauterine and newborn infections are generally more deleterious as opposed to those affecting adults, most probably due to the incomplete maturation of the BBB. Maturation of the innate and adaptive immune system is also an important aspect to consider [START_REF] Kollmann | Innate immune function by Toll-like receptors: distinct responses in newborns and the elderly[END_REF]. Moreover certain viruses selectively target dividing neuronal precursors. In a neuronal cell culture model of Alphavirus infection, it was shown for example that terminally differentiated, mature neurons express higher levels of immunoregulatory factors that help them mount a more rapid antiviral response in contrast to undifferentiated neurons (Schultz et al., 2015).

Mechanisms of Viral Entry in the CNS

Upon establishment of the infection at their entry site, most viruses spread locally in a cell-to-cell manner. After a first cycle of replication in target cells, the virus disseminates. Primary viremia -the presence of a virus in the blood -ensues. It is generally short-lived and of low titer and contributes to the spreading of the virus from peripheral tissues to other sites, such as to the lymphoid organs, leading to secondary viremia. This in turn lasts typically for longer periods of time and is higher in titer (Pekosz et al., 1995) (Figure I1.4). BBB. On the other hand, infection through peripheral nerves generally occurs upon binding of the virus to specific receptors found on axonal termini of peripheral neurons. Each of these mechanisms will be described in more details in the following sections.

One important aspect to consider when investigating mechanisms of viral entry in the CNS is the inoculation route used in experimental animal models. Certain viruses can in fact employ more than one mechanism; WNV, for example, has been shown to enter the CNS through infection of endothelial cells of the brain vasculature as well as through infection of olfactory neurons (Xu et al., 2012;[START_REF] Monath | Mode of entry of a neurotropic arbovirus into the central nervous system. Reinvestigation of an old controversy[END_REF]. Therefore, during sampling and analysis all cases should be taken into account and thoroughly investigated, to avoid missing important cues. polyomavirus [START_REF] Chapagain | Polyomavirus JC infects human brain microvascular endothelial cells independent of serotonin receptor 2A[END_REF], Epstein-Barr virus (Casiraghi et al., 2011), Human Cytomegalovirus [START_REF] Fish | Human cytomegalovirus persistently infects aortic endothelial cells[END_REF], and Mouse Adenovirus [START_REF] Gralinski | Mouse adenovirus type 1-induced breakdown of the blood-brain barrier[END_REF]. RNA viruses, such as WNV (Xu et al., 2012), Hepatitis C virus (Fletcher et al., 2012), and Human T cell leukemia virus (HTLV) [START_REF] Afonso | Alteration of blood-brain barrier integrity by retroviral infection[END_REF], have also been shown to affect BBB integrity.

In some cases, viruses can access the CNS by crossing areas where the BBB is not completely sealed. These include the choroid plexuses and the circumventricular organs 1 . Viruses can enter the CSF space and subsequently infect the underlying ependymal cells and surrounding brain tissues. Examples of such viruses are mumps virus [START_REF] Herndon | Ependymitis in Mumps virus meningitis: electron microscopic studies of cerebrospinal fluid[END_REF] and HIV (Falangola et al., 1995). The second possible mechanism through which viruses can gain access to the CNS is the so-called "Trojan horse" strategy. Circulating leukocytes such as monocytes/macrophages become infected and are able to release the virus in the CNS after infiltrating the brain parenchyma, where they normally act as immune sentinels. Once they reach the CNS, they shed infectious virions that establish CNS infection. Examples of such viruses include HIV and HTLV [START_REF] Kaul | Pathways to neuronal injury and apoptosis in HIV-associated dementia[END_REF]. JCV has also been shown to employ this mechanism, although it rather infects B cells that infiltrate the CNS of immune suppressed patients, causing a

1 Circumventricular Organs: Small structures bordering the subventricular spaces characterized by larger perivascular spaces and endothelial cells lacking tight junctions. They allow for greater exchange of fluids between the brain and the vasculature. From [START_REF] Johnson | Sensory circumventricular organs and brain homeostatic pathways[END_REF].

fatal brain disease [START_REF] Boothpur | Human Polyoma viruses and disease with emphasis on clinical BK and JC[END_REF]. Moreover, in a previous report it was shown that human macrophages become infected by SINV in vitro [START_REF] Assunção-Miranda | Proinflammatory response resulting from Sindbis virus infection of human macrophages: implications for the pathogenesis of viral arthritis[END_REF]. This has been however linked to the onset of arthritis in humans. To our knowledge, their contribution to SINV neuroinvasion in in vivo models has not yet been addressed.

Infection of Peripheral Neurons

Several pathogens infect and replicate in local nerve endings. These can either be sensory or motor neurons, extending outside the confinement of the CNS.

Nevertheless, they are connected to neurons of the CNS (through neurochemical synapses) and act as springboard for efficient entry and replication in the brain.

According to their specific receptor and entry route, neurotropic viruses will bind to either sensory or motor neurons and use anterograde and/or retrograde axonal transport systems to reach neurons in the CNS.

RV [START_REF] Ugolini | Rabies virus as a transneuronal tracer of neuronal connections[END_REF] and Poliovirus (PV) (Racaniello, 2006) are well-known examples of viruses that infect peripheral motor neurons. Subsequently, they use dynein-based components to retrogradely move along the axons up to the CNS.

On the other hand, herpesviruses use sensory-nerve endings as entry portal to the Peripheral Nervous System, where they maintain a quiescent state. Under certain conditions, such as stress stimuli, they can become reactivated and induce production of a large number of virus particles in a short amount of time [START_REF] Camarena | Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV-1 latency in neurons[END_REF] that can reach the CNS via retrograde transport.

Infection of the CNS can also occur from invasion of olfactory nerves, whose projections are found in the olfactory bulb of the CNS. Examples of such viruses include Influenza A virus, Herpes Simplex virus-1 (HSV-1), PV, Vesicular Stomatitis virus (VSV), RV, JEV, WNV, La Crosse virus [START_REF] Van Riel | The olfactory nerve: a shortcut for influenza and other viral diseases into the central nervous system[END_REF].

Munster et al. showed that Nipah virus infects the olfactory system of hamsters after intranasal inoculation [START_REF] Munster | Rapid Nipah virus entry into the central nervous system of hamsters via the olfactory route[END_REF]. CHIKV (Powers and Logue, 2007) and VEEV (Schafer et al., 2011) have also been suggested to use this entry path, in a model where mice were inoculated intranasally. Additionally, SINV was shown to propagate along peripheral nerves from the foot to the nose or the spinal cord prior to the brain of mice inoculated subcutaneously. This was determined by intravital imaging approaches using a luciferase-expressing recombinant SINV strain (Cook and Griffin, 2003). Anterograde transport uses the kinesin motor proteins, to move cargo from the soma to the synapse. HSV-1, for example, undergoes anterograde transport to exit via axonal varicosities at axon termini and infect neighboring cells [START_REF] Diefenbach | Transport and egress of Herpes Simplex virus in neurons[END_REF]. On the other hand, retrograde transport involves movement of cargo and viral particles from the synapse to the soma. This type of transport relies on proteins of the dynein family. RV interacts with the LC8 dynein light chain [START_REF] Raux | Interaction of the Rabies virus P protein with the LC8 dynein light chain[END_REF] and HSV-1 uses trans-synaptic release to reach connected neurons, while measles is thought to disseminate through microfusions between neighboring neurons [START_REF] Young | Making it to the synapse: Measles virus spread in and among neurons[END_REF].

Mechanisms of Spreading in the CNS

Localization

Antiviral Immune Responses in the CNS

The brain is generally considered an immunologically privileged site, as neuronal et al., 1994). Nevertheless, basal IFN activity seems to be low within the CNS, likely due to the toxicity of this cytokine to the brain (A detailed description of the IFN signaling pathway will be given in Chapter III).

Several studies have also shown the importance of molecules upstream or downstream of the IFN signaling pathways that become expressed in the CNS upon viral infection. In the case of upstream molecules, several Toll-like Receptors (TLRs) are widely expressed and have been shown to respond to neurotropic viruses [START_REF] Carty | Evaluating the role of Toll-like receptors in diseases of the central nervous system[END_REF]. Mutations in the endoplasmic reticulum UNC-93B protein (required for TLR3 signaling) [START_REF] Casrouge | Herpes simplex virus encephalitis in human UNC-93B deficiency[END_REF] and in TLR3 (Zhang et al., 2007) have been in fact associated with onset of Herpes Simplex Encephalitis. Downstream of IFN expression, Interferon Stimulated Genes (ISGs) have been found to be either specifically expressed in the CNS (e.g. ISG54 in the case of VSV) or in specific regions of the CNS (e.g.

Ifi27 and Rsad2/viperin/vig1, against WNV) [START_REF] Fensterl | Interferon-induced Ifit2/ISG54 protects mice from lethal VSV neuropathogenesis[END_REF][START_REF] Cho | Differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded RNA viruses[END_REF].

Depending on the virus, IFN can be produced by several cell types. In the case of La Crosse virus infection, for example, neurons are mostly responsible for IFN production [START_REF] Delhaye | Neurons produce type I interferon during viral encephalitis[END_REF], while Mouse Hepatitis virus induces IFN production mainly in macrophages and/or microglial cells [START_REF] Roth-Cross | Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia[END_REF]. are transcytosed to the endothelial lumen, where they are presented to attract circulating leukocytes [START_REF] Chang | Differential expression of TGF-β, IL-2, and other cytokines in the CNS of Theiler's murine encephalomyelitis virus-infected susceptible and resistant strains of mice[END_REF][START_REF] Middleton | Leukocyte extravasation: chemokine transport and presentation by the endothelium[END_REF]. Infiltration of leukocytes begins at late stages of infection. Cells normally accumulate in perivascular areas of the brain and later infiltrate the brain parenchyma in regions of virus infection, to subsequently induce the activation of the adaptive immune response. The peak of inflammatory cell accumulation generally occurs within two weeks after infection and, unless the virus persists, they are gradually eliminated [START_REF] Rowell | The inflammatory response to nonfatal Sindbis virus infection of the nervous system is more severe in SJL than in BALB/c mice and is associated with low levels of IL-4 mRNA and high levels of IL-10-producing CD4+ T cells[END_REF]. It has been shown, for example, in a mouse model of SINV infection that the virus can persist in the brain of mice that recovered from viral acute encephalitis for long periods of time in a non-productive form. Moreover, viral reactivation was associated with low levels of humoral immunity, suggesting the importance of antibody-mediated clearance [START_REF] Levine | Persistence of viral RNA in mouse brains after recovery from acute viral encephalitis[END_REF].

Summary of Chapter I

-The majority of EIDs are characterized by serious neurological manifestations and are mostly caused by zoonotic pathogens.

-Neurotropic arboviruses are kept in complex enzoonotic cycles but can sometimes escape, due to adaptation to a different vector. When humans or other vertebrates become infected, they are generally dead-end hosts.

-Neurotropic viruses often cause encephalitis, myelitis or meningitis.

-They can reach the CNS either via the bloodstream (by disrupting the BBB or by hijacking leukocytes) or via infection of peripheral nerves.

-Once in the CNS, they can spread via several mechanisms, including anterograde or retrograde transport along axons of infected neurons.

-Antiviral immune responses activated in the CNS differ from those in the periphery due to the non-renewable features of neurons. IFNs are the first cytokines to be expressed, in order to induce the antiviral state. Together with Rubiviruses, Alphaviruses are members of the Togaviridae family.

CHAPTER II: Alphaviruses -A Public Health Threat

As the etymology of the name suggests (from the Latin name for cape, toga), they are characterized by the presence of a lipid envelope, which is perfectly adherent to an icosahedral capsid, and containing a single-stranded positive-polarity RNA genome.

While the Rubivirus genus only contains the agent responsible for rubella, rubella virus, the Alphavirus genus is divided into seven antigenic complexes based on serological cross-reactivity and contains 29 recognized species (Powers et al., 2001) (Figure I2.1).

Depending on where they occur, they have been classified as either belonging to Neurovirulence of these viruses is associated with rapid and efficient spread throughout neurons of the Central Nervous System (CNS), resulting in death of infected neuronal cells [START_REF] Griffin | Alphaviruses[END_REF]. As can be expected, their pathogenicity, the global spread of the mosquito vectors as well as the lack of effective treatments and vaccines, make them a significant public health threat as both emerging infectious diseases and possible agents of bioterrorism [START_REF] Weaver | Host range, amplification and arboviral disease emergence[END_REF].

This introductory Chapter includes an overview of the molecular biology of

Alphaviruses. Subsequently, it focuses on several important aspects related to Old World Alphaviruses, including epidemiology, pathogenesis and immune responses. SINV, the pathogen used in this Thesis as a model to study viral-induced encephalitis in zebrafish embryos, is described in more details in the last section. Its importance for the study of viral-induced pathogenesis is well exemplified by the number of articles in which it appears as a model -over 60 articles published within the last year (e.g. ranging from studies on mechanisms of viral assembly and viral immune evasion, distinct immune responses, potential therapeutics). Nevertheless, many unanswered questions related to its neurotropism constitute the driving factor for the study presented in this Thesis.

Alphavirus Structure, Genome Organization and Replication Cycle

The virions of Alphaviruses are spherical and measure ~70 nm in diameter. They The genome is roughly 12 kb long, it includes a 5' terminal cap and a 3' terminal poly(A) tail, and it is divided into two Open Reading Frames (ORFs) separated by a well-conserved Untranslated Region (UTR) of ~120 nucleotides. Each ORF codes for multiple proteins generated by proteolytic cleavage. The 5' ORF, which is immediately translated upon virus entry, codes for four non-structural Polyproteins (nsP1-nsP4) required for transcription and replication. By contrast, the 3' ORF is not translated from the full genome, but from the subgenomic 26S RNA, which is made later in the viral cycle; it codes for the structural proteins (sPs): the capsid (C), the three envelope proteins (E3, E2, and E1) and a membrane-associated protein, 6K (Figure I2.2).

The Alphavirus nsP1 possesses both guanine-7-methyltransferase and guanylyl transferase activities, required for capping and methylation of newly synthesized genomes. nsP2 acts as triphosphatase and helicase on its N-terminal domain, and as protease for the nsPs on its C-terminal domain. nsP3 has been shown to act as phosphatase and is involved in modulation of pathogenicity in mice, while nsP4 is the RNA-dependent RNA-polymerase (RdRp).

While E3 is required for efficient particle assembly, E2 is known to be responsible for receptor binding as well as a determinant of neurovirulence, as demonstrated by studies on amino acid changes [START_REF] Ubol | Neurovirulent strains of Alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the E2 glycoprotein[END_REF]. 6K is essential for viral particle assembly at the plasma membrane and E1 is the Alphavirus fusion protein. The resulting nucleocapsid is transported to the cytoplasm, where the RNA is released after disassembly. Genomic RNA is used both for translation of proteins from the genomic and subgenomic RNA, as well as for transcription of nascent RNA. Lastly, the structural proteins are translated and encapsidate nascent genomic RNA before budding and release. Adapted from [START_REF] Leung | Replication of Alphaviruses: a review on the entry process of Alphaviruses into cells[END_REF].

Upon receptor-mediated binding to the cell surface and subsequent entry, viral particles undergo disassembly to release the genome into the cytoplasm (Figure I2. [START_REF] De Kinkelin | Interferon production in rainbow trout (Salmo gairdneri) experimentally infected with Egtved virus[END_REF]. The viral genome is then translated to generate the nsPs and sPs. After complete cleavage of the nsPs, they form a stable complex for the synthesis of negative strand antigenomes, then used as a template for synthesis of

Old World Alphaviruses: Epidemiology and Vector Distribution

Alphaviruses are kept in enzootic and epizootic cycles between blood-feeding arthropods and vertebrates. Vertebrate hosts can be divided into two groups: 1. the natural vertebrate host acting as the primary source of mosquito infection in which the virus replicates, but no symptomatic disease occurs and 2. vertebrate hosts that develop symptomatic disease.

For encephalitic Alphaviruses such as SINV, SFV, EEEV, and WEEV, the primary host is typically avian. In epizootic or epidemic cycles, infected mosquitoes transmit the virus to a secondary host, usually human or equine. Large outbreaks of disease occur when infected secondary hosts reach a high titer viremia, permitting further infection of mosquitoes and host populations.

Recurrent epidemics of both Old and New World Alphaviruses have been reported since the 20 th century. WEEV was the first encephalitic Alphavirus isolated from the brain of an infected horse [START_REF] Meyer | The etiology of epizootic encephalomyelitis of horses in the San Joaquin Valley[END_REF].

CHIKV was first isolated in 1953 in Tanzania, Africa and has since then caused frequent epidemics in Africa, Islands of the Indian Ocean, and Asia (Powers and Logue, 2007) together with the presence of the primary vector [START_REF] Jalava | Climatic, ecological and socioeconomic factors as predictors of Sindbis virus infections in Finland[END_REF]. This has been identified in members of the mosquito genera Culex and Culiseta. However also Aedes mosquitoes [START_REF] Lvov | Isolation of the causative agent of Karelian fever from Aedes sp. mosquitoes[END_REF] and, in a few cases, ticks have been found to carry and transmit the virus [START_REF] Gresikova | Identification of a Sindbis virus strain isolated from Hyaloma marginatum ticks in Sicily[END_REF].

A study conducted on the phylogeographic evolution of SINV has shown that over 50 different SINV strains exist [START_REF] Lundström | Phylogeographic structure and evolutionary history of Sindbis virus[END_REF] Their geographic distribution has been attributed to major bird migration patterns, validating the hypothesis that migrating birds are involved in large-scale viral dissemination [START_REF] Herrera | On the breeding distribution pattern of European migrant birds: MacArthur's theme re-examined[END_REF]. Moreover, SINV-associated disease outbreaks in South Africa, Sweden, Finland and Russia, are in accordance with the distribution of one of these five genotypes [START_REF] Brummer-Korvenkontio | Epidemiology of Sindbis virus infections in Finland 1981-96: possible factors explaining a peculiar disease pattern[END_REF].

Pathogenesis of Old World Alphaviruses

Old World Alphaviruses are generally associated with the development of long-lasting arthritis, as opposed to their New World counterparts, for which severe neuropathogenic diseases often result from the infection. After the 2005-2006 epidemic of CHIKV, however, there has been much debate as to whether Old World viruses can induce neurological symptoms, due to reports of severe cases of encephalitis, neuropathies, and deaths in infants and elderly patients [START_REF] Economopoulou | Atypical Chikungunya virus infections: clinical manifestations, mortality and risk factors for severe disease during the 2005-2006 outbreak on Reunion[END_REF]. Moreover, experimental models for other

Alphaviruses, such as SFV, RRV, EEEV and SINV, have shown similar neurological signs reminiscent of those observed in CHIKV infected patients [START_REF] Fazakerley | Virus tropism, distribution, persistence and pathology in the corpus callosum of the Semliki Forest virus-infected mouse brain: a novel system to study virus-oligodendrocyte interactions[END_REF][START_REF] Mims | Pathogenesis of Ross River virus infection in mice. I. Ependymal infection, cortical thinning, and hydrocephalus[END_REF][START_REF] Deresiewicz | Clinical and neuroradiographic manifestations of eastern equine encephalitis[END_REF]. Among these, SINV is the best characterized and has been widely used in mice to perform studies on neural pathogenesis of Alphaviruses, upon which it has been shown that it replicates in neural tissues causing encephalitis (Lewis et al., 1996;Cook and Griffin, 2003).

The disease progression in humans has been characterized as typically consisting of two phases: first, patients generally experience a mild, flu-like illness associated with replication at the primary site of infection, viremia, and initiation of the immune response. The second phase involves a second febrile episode directly linked with dissemination of the virus to other tissues [START_REF] Zacks | Encephalitic alphaviruses[END_REF]. During the acute phase of the disease, several symptoms other than fever can occur, such as headache, eye pain, generalized myalgia, arthralgia, diarrhea, vomiting, and rash [START_REF] Taubitz | Chikungunya fever in travelers: clinical presentation and course[END_REF]Powers and Logue, 2007).

During this phase, a number of patients develop neurological manifestations associated with mortality. Even though rare, Old World Alphavirus-induced neurological symptoms reported during the 2005-2006 CHIKV outbreak include encephalopathy associated with lymphopenia [START_REF] Lemant | Serious acute Chikungunya virus infection requiring intensive care during the Reunion Island outbreak in 2005-2006[END_REF], seizures, encephalitis, optic neuritis [START_REF] Mittal | Optic neuritis associated with Chikungunya virus infection in South India[END_REF], bilateral frontoparietal white matter lesions (which are considered as early signs of encephalitis), and lymphocyte infiltration in areas of demyelination and microglia activation in the gray matter with neuronal degeneration [START_REF] Ganesan | Chikungunya encephalomyeloradiculitis: report of 2 cases with neuroimaging and 1 case with autopsy findings[END_REF].

Moreover in some cases, dual infections of Alphaviruses with other endemic viruses can occur. Co-circulation of CHIKV and Dengue virus (DENV), for example, was reported in various States of India in 1964 and more recently in 2010. Data from the 2010 outbreak showed that many of the observed clinical symptoms were common in both monotypic and dual infections (Taraphdar et al., 2012). This not only increases the chances of a wrong diagnosis but also reduces the likelihood for the patient to reach a full recovery.

Role of Innate Immunity against Alphaviruses

Most of the data relative to the host response elicited from Alphavirus infections come from studies in small animal models and, in a few cases, non-human primates. It is general agreement that, upon onset of infection, type I Interferons (IFNs), mostly IFNα and IFNβ, are readily expressed and followed by production of virus-specific neutralizing antibodies at later time points.

The protective role played by type I IFNs against Alphaviruses has been demonstrated in several studies. Ryman et al. showed that adult mice lacking the IFN receptors (IFNAR -/-) infected with a neurovirulent SINV strain (NSV) succumb to the infection, as opposed to wild-type (WT) mice. Moreover, they were able to show that the lack of a fully functional IFN signaling pathway modifies the viral cell and tissue tropism and renders more cell types susceptible to the infection (Ryman et al., 2000). Very similar results have been obtained in the case of CHIKV infection. Couderc et al. showed in fact that IFNAR -/-adult mice are highly susceptible to CHIKV infection and that the clinical signs observed were in accordance with the cell types and tissues infected [START_REF] Couderc | A mouse model for Chikungunya: young age and inefficient type-I interferon signaling are risk factors for severe disease[END_REF]. Labadie et al. developed a cynomolgus macaque (Macaca fascicularis) model of CHIKV, in which infection was detected in several organs, such as in the liver, the spleen, the lymph nodes, the joints, the muscle, the skin and the meninges [START_REF] Labadie | Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages[END_REF]. Moreover, the authors showed that macrophages can act as a CHIKV reservoir and suggested that viral persistence accounts for the chronic CHIKV infection symptoms that are often observed in patients.

As can be expected, this group of viruses has developed evasive strategies to counteract the effects of IFNs. An in vitro study on SINV-infected cell cultures has shown that this role is played by nsP2 proteins, as they induce host cell transcription and translation shutoff [START_REF] Frolova | Roles of nonstructural protein nsP2 and alpha/beta interferons in determining the outcome of Sindbis virus infection[END_REF]. Studies performed on CHIKV-infected Human Embryonic Kidney-293 cell cultures have shown that the virus promotes activation of the autophagy machinery to enhance its own replication [START_REF] Krejbich-Trotot | Chikungunya virus mobilizes the apoptotic machinery to invade host cell defenses[END_REF]. In a comparative study between mouse and human cell cultures, it was shown that ubiquitinated capsids are bound to p62

(in both human and mice cells) and transported to the autophagolysosomes, thereby protecting infected cells from virus-induced death [START_REF] Judith | Species-specific impact of the autophagy machinery on Chikungunya virus infection[END_REF]. In contrast, human (but not mice) NDP52 interacts with nsP2 and favors viral replication. In the case of SINV, on the other hand, it was shown that autophagy-mediated clearance of SINV proteins is cytoprotective in neuronal cell types, via p62 and Atg5 (Overdahl and Levine, 2008).

Sindbis Virus

Sindbis virus, the prototypical Alphavirus, has been studied mainly in mice and Drosophila. In particular, studies in Drosophila have been directed towards a better understanding of RNA interference (RNAi) upon viral infections. Even though mosquitoes develop infection upon virus inoculation, RNAi has been shown to protect the arthropod from an otherwise lethal infection [START_REF] Cirimotich | Suppression of RNA interference increases Alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes[END_REF]. In fact, the combined action of cellular reverse-transcriptase activity and RNAi was shown to inhibit viral replication in favor of viral persistence [START_REF] Goic | RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila[END_REF].

On the other hand, the mouse model has helped in the characterization of virus-induced encephalitis. Despite its relatively low pathogenicity in humans, SINV is known to preferentially infect neurons and causes age-dependent encephalomyelitis in mice [START_REF] Tucker | Viral determinants of age-dependent virulence of Sindbis virus for mice[END_REF]. The increased survival of adult mice has been attributed, rather than to an incomplete maturation of the immune system, to a lower susceptibility of mature neurons to the virus. These have been found to express higher levels of IFN regulatory factors 3 (IRF3) and 7 (IRF7) molecules and to further induce them upon infection (Schultz et al., 2015). The genetic background of infected animal models has also been shown to determine different immune responses, which correlate with the different susceptibility observed between hosts [START_REF] Kulcsar | Distinct immune responses in resistant and susceptible strains of mice during neurovirulent alphavirus encephalomyelitis[END_REF].

SINV neurovirulence has been directly linked to specific amino acid changes in the E1 and E2 glycoproteins. In particular, Ala-72-Val and Asp-313-Gly of E1

(from the NSV to a less virulent strain) and His-55-Gln in E2, have been shown to considerably decrease the associated virulence of the strain (Lustig et al., 1988).

As a consequence, strains with specific E1 and E2 mutations have been shown to induce significant levels of apoptosis in brains and spinal cord of weanling but also of older mice (Lewis et al., 1996).

Studies on persistence of the virus after recovery from acute encephalitis have demonstrated that the virus can linger in a non-replicative form for long periods of time and reactivation is dependent on a decrease in the humoral response [START_REF] Levine | Persistence of viral RNA in mouse brains after recovery from acute viral encephalitis[END_REF]. The mechanism through which the virus can be cleared from the CNS is not fully understood, although it has been suggested to be dependent on tissue injury and neuronal cell death induced by infiltrating macrophages/microglia and T helper cells [START_REF] Kimura | Extensive immune-mediated hippocampal damage in mice surviving infection with neuroadapted Sindbis virus[END_REF].

One important aspect to consider when studying SINV-associated neurovirulence involves the viral strain being used. The comparison of different strains can provide important information not only into the genetic determinants that control the virulence phenotype but also into the observed differences in pathogenesis.

Different variants can exhibit great divergence in terms of organ, tissue and cell tropism as well as in the induced antiviral response. Brodie et al. showed that infection of mouse astrocytes with either a non-neurovirulent or with the NSV strains resulted in differential cell proliferation and expression of cytokines, which in the case of NSV were associated with changes characteristics of astrogliosis [START_REF] Brodie | Astrocyte activation by Sindbis virus: Expression of GFAP, cytokines, and adhesion molecules[END_REF].

Moreover, several SINV laboratory strains have been derived over the years.

These include for example the NSV strain used in the study of age-dependent virulence in mice [START_REF] Tucker | Viral determinants of age-dependent virulence of Sindbis virus for mice[END_REF] as well as recombinant strains, in which fluorescent reporter proteins have been inserted to follow various aspects of the infection process in vitro or ex vivo. SINV genome is in fact suitable for the insertion and consequent expression of genes of interest [START_REF] Geiss | Recombination-ready Sindbis replicon expression vectors for transgene expression[END_REF]. In particular, two SINV strains have been employed in this Thesis: 1.

pTE3'2J-eGFP, in which a second subgenomic promoter has been included at the 3' end for the insertion of specific genes of interest (i.e. GFP) (Hahn et al., 1992) and 2. pTR339-mCherry/2A, derived from the original African isolate AR339 [START_REF] Taylor | Sindbis virus: a newly recognized arthropod-transmitted virus[END_REF] and modified to include the mCherry reporter gene between the capsid and the E3 genes. Their differences in terms of genomic sequence and associated virulence have been addressed in Chapter II of the Results.

Summary of Chapter II

-Alphaviruses are single-stranded RNA enveloped viruses, transmitted to humans after the bite of an infected mosquito. They are normally divided into "arthritogenic" Old World Alphaviruses and "encephalitic" New World Alphaviruses, although several reports indicate that Old World viruses can sometimes induce neurological manifestations.

-Their genome is divided into two ORFs, coding for the non-structural proteins and structural proteins, respectively. Among the latter, several studies have identified specific amino-acid sequences of the E2 glycoprotein as determinants of neurovirulence.

-The geographical distribution of their vector is an important factor for the onset of an outbreak, as well as adaptation of the virus to new vectors.

-A functional IFN signaling pathway is essential for the survival of Alphavirus-infected hosts, although these viruses have evolved evasion mechanisms to subvert the immune response.

-SINV is encephalitogenic in mice and has therefore been employed as a model of Alphavirus-induced encephalitis.

CHAPTER III: Zebrafish -Host-Pathogen Interactions Unveiled

"Seeing is believing." John Clarke, from Paroemiologia Anglo-Latina ( 1639) "I wouldn't have seen it, if I hadn't believed it." Marshall McLuhan (1911-1980) The zebrafish (Danio rerio) is well established for the study of developmental biology in vertebrates. Studies of embryogenesis in the zebrafish date back to the 1930s and have been followed in the 1980s by works aimed at adapting this organism to forward genetic screens. It was thanks to the works of George

Streisinger and Charles Kimmel that the zebrafish became recognized as a valid vertebrate model for the study of gene function and human genetic disease [START_REF] Streisinger | Production of clones of homozygous diploid zebrafish (Brachydanio rerio)[END_REF][START_REF] Kimmel | A mutation that changes cell movement and cell fate in the zebrafish embryo[END_REF][START_REF] Lieschke | Animal models of human disease: zebrafish swim into view[END_REF].

Furthermore, the completion and release of the full zebrafish genome assembly in 2002 and its comparison to the human genome in 2013 (Howe et al., 2013) have greatly expanded the possibility of understanding the role of human genes for which no function has yet been assigned.

More recently, the zebrafish has emerged as an alternative vertebrate model to be used in a wide range of research fields including pharmacology and toxicology, cancer, neuroscience, and disease modeling [START_REF] Davis | Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos[END_REF][START_REF] Neely | Streptococcus-zebrafish model of bacterial pathogenesis[END_REF][START_REF] Phelan | Characterization of Snakehead rhabdovirus infection in zebrafish (Danio rerio)[END_REF]Ludwig et al., 2011). Its main experimental advantage, beyond its genetic tractability, consists in its transparency during early life that considerably facilitates in vivo imaging. It is therefore relatively easy to follow the distribution of fluorescent-protein-tagged pathogens in the whole organism in real time. At the same time, small regions with subcellular details can be visualized with modern confocal microscopy techniques and very little invasiveness (Palha et al., 2013).

Its small size, large number of eggs per clutch, and relatively low husbandry costs are also important factors that make zebrafish a valuable model organism. The following Chapter is therefore dedicated to some general aspect of zebrafish biology, with a particular focus on its Central Nervous System (CNS)

development. Subsequently, it discusses some important aspects regarding the development of the zebrafish immune system. Finally, it gives a brief overview of the advantages and disadvantages of the model.

Zebrafish -General Aspects on Biology and Development

The zebrafish belongs to the family Cyprinidae, infraclass Teleostei, class

Actinopterygii. Teleost fish constitute a monophyletic, very successful group of vertebrates with more than 20,000 species, that likely originated 350 million years ago and underwent an additional event of whole-genome duplication, called teleost-specific genome duplication [START_REF] Meyer | Gene and genome duplications in vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions[END_REF].

Zebrafish is a tropical freshwater fish. It is indigenous to the south-eastern Himalayan region and is commonly found in India, Pakistan, Bangladesh, Nepal and Burma. In the wild, zebrafish are thought to be a largely annual species, although when kept in captivity they can survive up to 3.5-5 years. They normally feed on zooplankton and insects, and can breed all year round, although the main period of reproduction occurs just before the onset of the monsoon season [START_REF] Spence | Diet, growth and recruitment of wild zebrafish in Bangladesh[END_REF]. When kept in captivity, they can easily be led into mating by simulating night/day circadian cycles, with light onset being the trigger. This has become common practice in zebrafish facilities worldwide. and 32°C and the optimal temperature is 28.5°C (83.3°F), used as the reference for staging [START_REF] Kimmel | A mutation that changes cell movement and cell fate in the zebrafish embryo[END_REF]. After 3 dpf, zebrafish are generally referred to as larvae; the swim bladder inflates shortly after and by 4-5 dpf, they exhibit autonomous feeding behaviour. At around 24 dpf, zebrafish are considered juvenile (coinciding with the development of scales) and reach sexual maturity around 3-5 months, depending on the environmental conditions, after which are considered adults [START_REF] Parichy | Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish[END_REF] (Figure I3.1).

Zebrafish Central Nervous System: Organization and Development

The small size and transparency of the zebrafish larva make it an attractive model to study the formation and function of the vertebrate brain. The fast-growing importance of the zebrafish for the neuroscience field is well exemplified by the increasing number of available bioinformatic resources. Recently, Ullmann et al.

have developed a three-dimensional digital atlas of the zebrafish brain, which provides detailed spatial information of all neuronal structures [START_REF] Ullmann | A three-dimensional digital atlas of the zebrafish brain[END_REF]. Researchers from University College London and King's College London, in the United Kingdom, have launched zebrafishbrain.org, a website with updated neuroanatomical data and resources for the zebrafish research community. Ahrens et al. showed that light-sheet microscopy is a powerful tool to detect single-cell neuronal activity while imaging the whole brain in vivo [START_REF] Ahrens | Whole-brain functional imaging at cellular resolution using light-sheet microscopy[END_REF].

These studies, together with the work performed by Wullimann et al., greatly contributed to our understanding of the zebrafish brain, its organization, and its development (Wullimann et al., 1996).

Zebrafish Brain Anatomy

The zebrafish brain is very similar in its general structures to that of higher animals and can be divided into five regions: the telencephalon, the diencephalons, the mesencephalon, the metencephalon, and the myelencephalon [START_REF] Speare | Nervous system[END_REF][START_REF] Roberts | The anatomy and physiology of teleosts[END_REF]) (Figure I3.3).

Together, the telencephalon and the diencephalon constitute the forebrain, the most anterior region of the brain. The midbrain or mesencephalon is the brain region between forebrain and hindbrain. Finally, the hindbrain, which includes the metencephalon and the myelencephalon, is the most posterior region of the brain. -The telencephalon controls olfaction, memory, reproductive behavior, feeding behavior, and color vision. The olfactory organ is directly connected to the telencephalon via the olfactory bulb.

-The diencephalon can be subdivided into three components: the epithalamus, the thalamus, and the hypothalamus. The thalamus and the hypothalamus act as correlation centers for sensory inputs such as olfaction and gestation; however, the complete function of the thalamic region has not yet been identified. Several features of the mammalian thalamus, such as connection to the isocortex, are absent in zebrafish and more studies are needed in order to better define evolutionary differences [START_REF] Mounce | Inhibition of polyamine biosynthesis is a broad spectrum strategy against RNA viruses[END_REF].

-The mesencephalon is anatomically subdivided into the optic tectum, at the roof, and the tegmentum, at the floor. The optic tectum is relatively large and is divided longitudinally into two globular structures -the corpora bigemina -involved in the reception and the coordination of optic nerve inputs. The zebrafish eye is similar to that of all other vertebrates and consists of three regions: 1. the tunica fibrosa, which encompasses the cornea and the lens 2. the tunica vasculosa, 3. the retina. In the retina, several layers can be distinguished: a. the pigment epithelium, which contains photoreceptor cells, b. the external nuclear layer, with the nuclei of photoreceptor cells, c. the bipolar cells that connect photoreceptor cells to d. the retinal ganglion cells (RGCs). These send their axons through the optic fibers into the optic nerve [START_REF] Diekmann | Characterization of optic nerve regeneration using transgenic zebrafish[END_REF].

-The cerebellum, or metencephalon, is associated with reception and coordination of proprioceptive and balance stimuli.

-The myelencephalon is the most posterior region of the brain, from which the medulla oblongata develops. The medulla connects the higher regions of the brain to the spinal cord.

Zebrafish Neuronal and BBB Development

The CNS of vertebrates develops from the neuroectoderm, a specialized region of the ectoderm that matures at the end of the gastrulation phase (~10 hpf in zebrafish embryos). Neurulation in zebrafish resembles the secondary neurulation observed in higher vertebrates, characterized by condensation of the mesenchyme to form a rod, which then undergoes an epithelial transition from the neural plate into the neural tube4 (Figure I3.3).

In zebrafish, the first neurons become post-mitotic in the neural plate shortly after gastrulation; therefore embryos start twitching as soon as 1 dpf [START_REF] Eisen | Pathway selection by growth-cones of identified motoneurons in live zebrafish embryos[END_REF]. At 2 dpf, they start to respond to touch, thanks to the action of a few differentiated neurons and a simple scaffold of axons [START_REF] Kimmel | Patterning of the brain of the zebrafish embryo[END_REF]. Early neurons are distributed in three longitudinal columns of the neural plate.

Within these columns, only a subset of cells express the HuC marker, typical of differentiated neurons. Expression of the neurogenin1 (NGN1) transcription factor helps define the longitudinal proneuronal domains where cells have the potential to become neurons [START_REF] Korzh | Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation[END_REF]. However, only a subset of cells maintains high levels of NGN1 [START_REF] Haddon | Multiple delta genes and lateral inhibition in zebrafish primary neurogenesis[END_REF] Da) start to be retained from 5 to 10 dpf, time at which the BBB is fully formed (Fleming et al., 2013).

Zebrafish Antiviral Innate Immune System

The following section is an extract from: "The Antiviral Innate Immune Response in Fish: Evolution and Conservation of the IFN System" (J. Mol. Biol., 2013).

This review article represents a collaborative effort between the two groups in which I did my Thesis work. The full article is included at the end of this Thesis in

Annex 1.

Interferons (IFNs) represent the first line of defense against viral infections. Their vital role is testified by the conservation of their structure among vertebrates (Hamming et al., 2011). IFNs belong to the class II helical cytokine family and, in mammals, can be divided into three different groups based on biological and structural features as well as receptor usage (Pestka et al., 2004): mammalian

IFNs have been classified as type I (α, β, ω, ε, and κ), type II (γ), and type III (λ)

IFNs. Actually, only type I and type III IFNs (often grouped under the label "virus-induced IFNs") are truly specialized as innate antiviral cytokines; IFNγ is rather a regulatory cytokine of innate and adaptive immunity, mostly active against intracellular bacteria.

Zebrafish possess four IFN genes (IFNφ1-4) structurally similar to mammalian type I IFNs (Zou and Secombes, 2011;Aggad et al., 2009). This number is unlikely going to change considering the quality reached by the zebrafish genome assembly.

At the cellular level, macrophages and neutrophils represent the first immune cell types activated at the larval stage in response to pathogens. In fact, it was shown that by 3 dpf the innate immune system is fully functional, with macrophages being able to phagocytose bacteria as early as 30 hpf [START_REF] Herbomel | Ontogeny and behaviour of early macrophages in the zebrafish embryo[END_REF] and neutrophils from 52 hpf (Le [START_REF] Guyader | Origins and unconventional behavior of neutrophils in developing zebrafish[END_REF]. Moreover, in the case of CHIKV infection in zebrafish, neutrophils (together with hepatocytes) were

shown to be the main IFN-producing cell type (Palha et al., 2013).

Upstream IFN Signaling Pathway

The activation of the IFN system is dependent on the specific recognition of conserved pathogenic motifs -known as PAMPS, for pathogen-associated molecular patterns -by the numerous pathogen-recognition receptors (PRRs)

found on intracellular vesicles [START_REF] Kawai | Toll-like receptors and their crosstalk with other innate receptors in infection and immunity[END_REF]. Antiviral PRRs include for example RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs). In silico analyses have revealed that they are highly conserved between mammals and teleost fish (Rajendran et al., 2012). Sequence analyses have also suggested a fair conservation of signaling pathways downstream of RLRs, with a critical role played by the mitochondrial antiviral signaling protein (MAVS) (Zou et al., 2009;Biacchesi et al., 2009;Simora et al., 2010;Su et al., 2011, Xiang et al., 2011).

Association of MAVS with TRAF3 (tumor necrosis factor (TNF)

receptor-associated factor 3) and activation of the pathway by TBK1 (TANK binding kinase 1) via phosphorylation of IFN regulatory factor 3 (IRF3) and 7

(IRF7) transcriptional factors have also been shown in fish (Xiang et al., 2011;Sun et al., 2010). Nuclear translocation of these factors induces the transcription of different cytokines including IFN genes (Figure I3.5).

Downstream IFN Signaling Pathway

In zebrafish, the two groups of IFNs signal via two different receptors. IFNφs of the first group (IFNφ1 and φ4) bind to the cytokine receptor family B (CRFB)

CRFB1-CRFB5 complex while the receptor for group II (IFNφ2 and φ3) consists of the association between the CRFB5 and CRFB2 chains (Levraud et al., 2007;Aggad et al., 2009) (Figure I3.6).

In mammals, IFN binding to their membrane receptors (IFNAR) triggers recruitment and binding of the kinases TYK2 and JAK1 to IFNAR1 and IFNAR2, respectively. Subsequently, these kinases promote the phosphorylation of STAT1 and STAT2 proteins preceding their oligomerization. Conjugation of cytoplasmic IRF9 to the STAT1/2 oligomers generates the complex ISGF3 (IFN-stimulated gene factor 3), which induces the transcription of IFN stimulated genes (ISGs)

after binding nuclear IFN-stimulated responses elements on their promoter. The zebrafish genome encodes two different paralogues, stat1a and stat1b (Stein et al., 2007). The kinases JAK1 and TYK2 as well as STAT2 and IRF9 are also present in fish genomes (Stein et al., 2007). Type I IFNs do not possess antiviral activity per se but rather interfere with viral infection through induction of a vast repertoire of ISGs via the JAK/STAT pathway. A few hundred ISGs have been identified in humans (Sadler and Williams, 2008;Schoggins et al., 2011), with a rich diversity of molecular functions. Some ISGs exert a direct antiviral activity such as MX, RSAD2/VIPERIN/VIG1, ISG15, PKR, and TRIM5. However, the connection of most ISGs to antiviral mechanisms, and even their role in the biology of the cell, remain unknown.

While ISGs are intrinsically located downstream of IFN in the antiviral pathways induced by viral infections, a number of them are able to up-regulate type I IFNs and are therefore involved in positive feedback regulatory loops (e.g. RSAD2) (Saitoh et al., 2011), while some also feedback negatively on IFN signaling (e.g. SOCS1 and 2). Furthermore, the recognition of viral compounds by cellular sensors can up-regulate some ISGs directly, that is, independently of IFN induction; such bypass has been shown for example for MX (Goetschy et al., 1989;DeWitte-Orr et al., 2007) and for RSAD2 in humans and fish (Zhu et al., 1997;Boudinot et al., 1999).

In a recent study performed by our lab, zebrafish larvae were shown to possess ~100 ISGs orthologs to human ISGs [START_REF] Briolat | Contrasted innate responses to two viruses in zebrafish: Insights into the ancestral repertoire of vertebrate IFN-stimulated genes[END_REF], although their function has yet to be characterized. In this comparative study between the effects of Infectious Hematopoietic Necrosis virus (IHNV) and Chikungunya virus (CHIKV) on ISGs induction, it was shown that ISGs followed the same kinetics of IFNs while being expressed in a tissue-specific manner. Moreover, several were induced by CHIKV and repressed by IHNV, suggesting the presence of antagonist pathways.

Models of Viral Infections in the Zebrafish

Free-swimming larvae, which have to rely on innate immune defenses before maturation of adaptive immunity, are highly suited for the study of innate immunity and inflammation; as previously described, the overall similarity of the cytokine networks between fish and mammals as well as their immune cells [START_REF] Traver | The zebrafish as a model organism to study development of the immune system[END_REF] make their use relevant for many diseases. Moreover, characterization of IFNs and ISGs pathways in zebrafish larvae has been the focus of the aforementioned study, performed in our labs [START_REF] Briolat | Contrasted innate responses to two viruses in zebrafish: Insights into the ancestral repertoire of vertebrate IFN-stimulated genes[END_REF]. This has greatly contributed to our understanding of viral-specific immune responses and provides a useful reference for studies of host-virus interactions.

Several human viruses have already been studied in zebrafish, such as Hepatitis B and C viruses [START_REF] Liu | A zebrafish model of intrahepatic cholangiocarcinoma by dual expression of hepatitis B virus X and hepatitis C virus core protein in liver[END_REF][START_REF] Ding | Zebrafish as a potential model organism for drug test against Hepatitis C virus[END_REF], Herpes simplex virus-1

(HSV-1) [START_REF] Antoine | Zebrafish: modeling for Herpes Simplex virus infections[END_REF], and recently by our lab, CHIKV (Palha et al., 2013). Fish virus models include for example Viral Hemorrhagic Septicemia virus [START_REF] Novoa | Zebrafish (Danio rerio) as a model for the study of vaccination against viral haemorrhagic septicemia virus (VHSV)[END_REF], Spring Viremia of Carp virus [START_REF] Encinas | Identification of multipath genes differentially expressed in pathway-targeted microarrays in zebrafish infected and surviving Spring Viremia Carp virus (SVCV) suggest preventive drug candidates[END_REF], and IHNV (Ludwig et al., 2011).

Moreover, a variety of tools including various transgenic reporter fluorescent lines have been developed to follow inflammatory cell types and responses in zebrafish.

Among these, several were employed for this Thesis to follow in vivo the activity of macrophages (mpeg1:Gal4/UAS:NfsB-mCherry transgenic larvae [START_REF] Ellett | Mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish[END_REF]), neutrophils (LyZC:DsRed larvae [START_REF] Hall | The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish[END_REF]) and IFN-producing cells (ifnφ1:mCherry larvae (Palha et al., 2013)) in the context of SINV infection.

Concluding Remarks

Several factors have contributed to the considerable expansion of the zebrafish as an animal model in medical research, with its small size, its transparency, and the high similarity of its immune system to that of mammals being only a few [START_REF] Renshaw | A model 450 million years in the making: zebrafish and vertebrate immunity[END_REF].

Thanks to the complete annotation of the zebrafish genome, several reverse genetics techniques have become available, such as retrovirus-mediated insertional mutagenesis [START_REF] Golling | Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development[END_REF], mutant identification using Targeting Induced Local Lesions In Genomes (TILLING) technology [START_REF] Wienholds | Target-selected inactivation of the zebrafish rag1 gene[END_REF] or the most recent one, CRISPR/Cas9-mediated knock-in approach for genome editing [START_REF] Hwang | Efficient genome editing in zebrafish using a CRISPR-Cas system[END_REF][START_REF] Auer | CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish[END_REF]. As previously mentioned, generation of new transgenic reporter lines has almost become routine, and gene knock-down is made easy by microinjection of antisense Morpholino Oligonucleotides (MOs) at the 1 to 4 cell stage. Zebrafish are also regularly used in chemical screens, since they allow for testing of a large number of compounds in vivo by simply adding these into the water [START_REF] Baker | Screening: the age of fishes[END_REF].

However, this model presents a few disadvantages as well. The maximum temperature at which zebrafish can be raised is 32°C (89.6°F), which impairs the study of those pathogens that replicate at higher temperatures. The standard laboratory zebrafish lines are not fully inbred and the genetic background of mutants and transgenic lines is generally poorly characterized. There is a limited amount of available biochemical tools (i.e. antibodies) or of established in vitro cell lines, and it is extremely challenging to establish cell cultures. As a matter of fact, part of this Thesis has consisted in the development of a primary neuronal cell culture system from zebrafish brain homogenates. The work is still in its infancy but preliminary data have shown that this could be a valuable new tool to address questions such as neuronal survival and gene expression changes in response to pathogens.

Summary of Chapter III

-Zebrafish is a tropical freshwater fish, originally employed for studies of embryogenesis and more recently also in cancer, neuroscience, pharmacology, and disease modeling fields.

-Its main advantage lies in the optical clarity of its embryos and larvae.

Transgenic lines expressing fluorescent reporter proteins in cells or tissues of interest are now being developed routinely.

-The zebrafish brain consists of five major regions: the telencephalon, the diencephalons, the mesencephalon, the metencephalon, and the myelencephalon, each one well-characterized anatomically, although functional knowledge is unequal.

-The zebrafish antiviral immune system shares overall similarity with the human one, relying heavily on the expression of IFNs and ISGs.

-The replication, tropism, and pathogenesis of several human and fish viruses have been characterized and/or are being currently characterized.

-Despite a few disadvantages, the zebrafish is a very powerful model to visualize in real time at the whole-organism level events taking place at the cellular or sub-cellular level.

AIM OF THE WORK

The laboratories where I worked during my PhD Thesis previously pioneered the A notable finding in the study of the model of CHIKV infection in zebrafish was the entry of the virus in the brain, despite the fact that the virus is inoculated in the bloodstream at a stage where the blood-brain barrier is mature enough to prevent passive passage of proteins. Remarkably, this virus was then shown to persist in the brain much longer than in other tissues. Moreover, neutrophils appeared to be the main cell population activated in zebrafish larvae to clear the infection but they were excluded from the brain, suggesting other mechanisms are involved in the control of the infection in this particularly sensitive organ (Palha et al., 2013).

These results laid the foundation for my project.

The aim was in fact to gain better insights into the mechanism through which

Alphaviruses persist longer in the CNS than in other infected organs. To do so, Sindbis virus was the obvious choice. The virus is markedly neurotropic in mice but is nevertheless regarded as a BSL2 pathogen, and as such allowed us to use our confocal microscopy equipment for both ex vivo and in vivo imaging, unlike CHIKV, which is classified as a BSL3 pathogen.

We therefore proceeded to characterize the general course of SINV infection in zebrafish larvae, with a special attention towards its possible mechanism of entry in the CNS. The data collected on this part of the project constitute the submitted article included in the first part of the Results section.

At the same time, we surveyed the IFN-related immune response, from its onset to later times post-infection. The results are still very preliminary but indicate that depending on the level of brain infection -mild in the case of CHIKV and extended in the case of SINV -different responses may be activated in the CNS.

This differential immune response has also been observed after the production and comparison of different SINV strains (2 nd part of the Results section).

Part of this Thesis project has also been devoted to the development of a protocol to obtain neuronal cell cultures from zebrafish larvae, with the aim to determine the mean survival rate of isolated neurons and glial cells upon SINV or CHIKV infection and to possibly visualize the spreading mechanism within infected neurons (anterograde and/or retrograde movement).

Finally, we set up a collaboration with the group "Viral populations and Pathogenesis", led by Marco Vignuzzi at Pasteur Institute, with the aim to exploit our SINV infection model to test potentially antiviral compounds.

RESULTS

RESULTS

The results obtained during this Thesis have led to the characterization of the infection course of Sindbis virus (SINV) in zebrafish larvae, with a special attention towards its neurotropism. Thanks to the combined use of a green fluorescent protein (GFP)-recombinant SINV strain (SINV-GFP) and zebrafish transgenic larvae, we have been able to follow in vivo and at cellular resolution SINV infection course as well as the elicited immune response. Data collected on SINV mechanism of entry in the Central Nervous System (CNS) have been included in the submitted paper and constitute the 1 st Chapter of this section.

The 2 nd Chapter of the Results involves the studies we performed on the differential immune responses observed in the periphery and in the CNS.

Moreover, we have produced a second recombinant SINV strain (SINV2-mCherry) and have used it to characterize modulations in the antiviral Interferon (IFN)-mediated response by different virus strains. We have also set up a protocol to obtain zebrafish neuronal and glial cells in culture (Chapter 3). These will represent a valuable tool for the growing zebrafish research community, to study mechanisms of pathogenesis and cell survival under several stimuli, such as viral infections.

The final Chapter of the Results includes preliminary data on the potential antiviral properties of difluoromethylornithine (DFMO) on the pathogenicity of SINV. These in turn could be useful in the future to screen for and identify other potential broad-spectrum antiviral drugs.

Introduction

The outcome of an infection critically depends on the organs reached by the invasive pathogen, which entails a precarious balance between the pathogen dissemination strategy and systemic and local host defenses. The central nervous system (CNS) is especially vulnerable, being highly susceptible to both virus induced cytopathic effects and to the inflammatory response itself [START_REF] Swanson | Viral diseases of the central nervous system[END_REF]. The CNS is however protected by specialized barriers, notably the blood-brain barrier (BBB). Neuroinvasion is generally rare but some viruses have evolved strategies to enter and spread within the CNS. Rabies virus is a well-known example of a neuroinvasive -and fatal -pathogen in nearly 100% of human cases [START_REF] Dietzschold | Concepts in the pathogenesis of rabies[END_REF]. Poliovirus can reach neurons in the spinal cord, brain stem or motor cortex, inducing characteristic paralysis, but only in 1-2% of infected individuals (Racaniello, 2006). Herpesviruses reach the peripheral nervous system and establish a lifelong persistent infection. While generally quiescent, they can revert under certain stress stimuli to produce infectious particles and in rare cases reach the CNS [START_REF] Tirabassi | Molecular mechanisms of neurotropic herpesvirus invasion and spread in the CNS[END_REF]. Persistent or "stable" infection of the CNS is also a rare occurrence, except for some specialized viruses. On the other hand, acute CNS infections are more often caused by zoonotic pathogens, for which humans normally represent a "dead-end host" (Gubler, 2001). In particular, among the arboviruses -viruses transmitted by arthropod vectors -Alphaviruses constitute a major source of viral zoonotic diseases, and often induce encephalitis. Eastern (EEEV) and Western Equine Encephalitis virus (WEEV) but also Chikungunya virus (CHIKV) have been associated with serious neurological manifestations.

Despite extensive studies in the field, the factors that determine the onset of neuroinvasion are not well understood. Infants are clearly more prone to CNS invasion than adults, generally attributed to an immature BBB. Among adults, genetic factors have been identified in a few cases. Mutations in the ccr5 gene (a chemokine receptor and co-receptor for Human Immunodeficiency Virus -HIV), for example, have been shown to increase the incidence of symptomatic West Nile virus (WNV) infection (Glass et al., 2006). With respect to cellular determinants, maturation of neurons has been shown to impair viral replication of certain arboviruses in contrast to non-differentiated neurons (Schultz et al., 2015).

As of today, some studies have addressed ways to limit neurological symptoms of encephalitis (e.g. [START_REF] Brison | Novel treatment with neuroprotective and antiviral properties against a neuroinvasive human respiratory virus[END_REF] but no strategy exists to specifically prevent CNS invasion during outbreaks of neurotropic viruses.

The mechanisms of entry of viruses in the CNS have been characterized using a variety of approaches both in vitro and ex vivo, and fall within three categories.

Some viruses are able to infect cells of the BBB (i.e. endothelial cells that form brain microvessels) or other components of the brain-periphery interface, such as epithelial cells of choroid plexuses. This allows their release in the brain parenchyma by the infected cells and/or disruption of the barrier causing leakage of blood-borne virions. Another strategy is known as the "Trojan horse" entry:

viruses hide in monocytes/macrophages and exploit the ability of these cells to cross the barrier to enter the brain. Finally, various viruses travel within the numerous axons of motor or sensory neurons that connect the CNS to the periphery, using either anterograde or retrograde transport [START_REF] Koyuncu | Virus infections in the nervous system[END_REF].

Despite the development of virus mutants built to express reporter genes (e.g. luciferase, GFP, mCherry), intravital imaging of virus infection in mammals is still a real challenge [START_REF] Rameix-Welti | Visualizing the replication of Respiratory Syncytial virus in cells and in living mice[END_REF]. A major hindrance in detailed kinetic analyses of CNS viral invasion lies with observing these events in real time. The zebrafish larva offers a powerful system to follow such events in a vertebrate, being small, transparent, and tolerant for long anesthesia (Levraud et al., 2014). In a recent study, we performed real time imaging of zebrafish larvae inoculated with a green fluorescent protein (GFP) recombinant CHIKV (Palha et al., 2013). CHIKV is a re-emerging arbovirus, which has gained attention after the epidemic of 2005-2006 in Islands of the Indian Ocean (Powers and Logue, 2007) and, more recently, due to its spread to the Caribbean, the United States and

Polynesia. In our previous study in zebrafish larvae, the majority of infected cells was found in various peripheral organs; however, infection of some brain cells always occurred. Remarkably, infected cells in the CNS survived much longer than cells infected in the periphery. Importantly, those experiments were performed at a developmental stage where the BBB of the zebrafish larva is mature enough to block the passage of proteins from the blood to the CNS (Fleming et al., 2013), excluding passive diffusion of viruses to the CNS. This work demonstrated that it is possible to image neuroinvasion in zebrafish larvae from the earliest stages of the infection. However the BSL3 status of CHIKV limited our practical options for in vivo microscopy.

To better characterize Alphavirus neuroinvasiveness, we decided to employ in this work Sindbis virus (SINV), the Alphaviridae prototype and a BSL2 pathogen markedly neurotropic in mice (Lewis et al., 1996;Lustig et al., 1988). SINV has often been used for studies of viral spreading in the CNS after intracerebral (ic) inoculation, rather than to understand its mechanism of neuroinvasion. However, its entry into the CNS has been suggested to occur from infection of peripheral nerves (Cook and Griffin, 2003), although the resolution achievable in mice did not permit to distinguish details below tissue or organ level, and alternative hypotheses were not tested.

Here, we show that upon intravenous (iv) inoculation in zebrafish larvae SINV infects various cell types and replicates efficiently. Moreover, in a substantial fraction of infected larvae the virus reaches the CNS, where its progressive spread can be documented by in vivo imaging. We tested the different possible routes of invasion of the CNS and identified axonal transport via infected peripheral neurons as the most likely route of entry. In addition, we followed the elicited host response and highlighted the importance of type I interferons (IFN) in preventing spreading of the virus in the periphery, but not for neuroinvasion.

Results

SINV shows a broad organ tropism in infected zebrafish larvae

Wild type (WT) zebrafish larvae were inoculated intravenously (iv) at 3 days post-fertilization (dpf) (Figure 1A) with ~10 2 TCID 50 of a recombinant SINV strain: SINV-GFP (hereon referred to as SINV), engineered to induce GFP expression in infected cells (Hahn et al., 1992). Viral growth was determined by qRT-PCR of the viral E1 gene from whole larvae lysates (Figure 1B). Intense viral replication occurred during the first 48 hours of the infection and subsequently leveled off and declined over time.

We observed infected larvae with a dissecting stereomicroscope; relatively mild and usually transient signs of disease appeared as early as 2 days post-inoculation (dpi), including edema, irregular heartbeat, and yolk opacification. In a minority of larvae (~15%), these signs persisted and led to death around 4 dpi (Figure 1C).

Live fluorescence microscopy revealed the presence of GFP-positive (GFP + ) cells scattered or in clusters. These infected cells were mostly observed in peripheral organs including the liver, the heart and the yolk (Figure 1D). In addition, the infection often reached the CNS (Figure 1E).

Fixation and whole-mount immunohistochemistry (WIHC) with anti-GFP and anti-SINV capsid antibodies confirmed that GFP + cells were infected and actively undergoing viral protein translation and assembly (Figure S1A. Related to Figure 1).

These results indicate that SINV replicates efficiently in zebrafish larvae and that its spread through the whole organism can be monitored over time via in vivo imaging (Figure 1; Figure S1).

SINV is neuroinvasive in zebrafish larvae

Under the fluorescence microscope, a prominent feature of a number of SINV inoculated larvae was the presence of GFP + cells in the brain or the spinal cord (Figure 1E). Approximately half of the larvae had obvious CNS infection by 3 to 7 dpi, although this was less prominent at early time-points. The course of the infection was therefore evaluated every 24 hours by following individual larvae with confocal imaging during several consecutive days. Unlike infection foci in the periphery, those observed in the brain were either persisting or progressively increasing over time (Figure 2A). Co-immunolabeling to stain SINV-positive cells and axonal projections confirmed that CNS neurons were infected (Figure 2B).

Peripheral neurons, such as trigeminal ganglion cells and retinal ganglion cells, were also targeted by the virus (Figure 2C; Figure S2A. Related to Figure 2).

Although the pattern of brain infection was very variable between individuals, analysis of a large panel of samples revealed that infection foci were commonly found in the olfactory bulb, the optic tectum, the vestibulolateralis lobe of the corpus cerebelli and the medulla (Figure S2B and S2C. Related to Figure 2).

To follow infection of neurons in vivo, we used elavl3:Gal4/5xUAS:RFP doubletransgenic larvae (from hereon called HuC:G/U:RFP), in which RFP is expressed in postmitotic neurons. Observations from early stages of infection (~8 hpi)

showed that the first infected cells to appear after iv injection were in the periphery (e.g. muscle cells, cells in the heart and in the liver), followed at later time-points by neurons of the peripheral and the central nervous system (1dpi) (Figure 2D).

To exclude the possibility that neurons may simply require longer to express GFP once infected, we performed intracranial (ic) inoculation of SINV in HuC:G/U:RFP larvae (Figure 1A). GFP + neurons were detected as soon as 8 hpi (Figure 2D), indicating that late appearance of CNS infection in iv injected larvae is a result of late infection of neurons. Ic injection resulted in a more severe disease with over 50% of larvae dying between 5 and 7 dpi (Figure S2D. Related to Figure 2). Viral growth kinetics measured by viral transcripts from whole larvae homogenates was slightly higher in ic injected larvae than in iv inoculated ones (Figure S2E. Related to Figure 2). Many foci of GFP + cells were systematically found in various regions of the brain and spinal cord, but little spreading occurred at the periphery (e.g. eye) (Figure 2E).

Overall, these results show that after inoculation of the virus in the bloodstream, peripheral cells are infected first, followed by invasion of the CNS (Figure 2;

Figure S2).

SINV does not target the blood-brain barrier endothelium

We then proceeded to test the possible mechanism of SINV entry in the CNS. A number of DNA and RNA viruses have been shown to gain access through infection of endothelial cells of the BBB, including for example Epstein-Barr virus (Casiraghi et al., 2011), Hepatitis C virus (Fletcher et al., 2012), and WNV (Xu et al., 2012). Moreover, our laboratory previously established a zebrafish infection model of a fish rhabdovirus, Infectious Hematopoietic Necrosis Virus (IHNV), in which we demonstrated that IHNV primarily infects endothelial cells of the vasculature. Infection of these cells in turn causes a rapid disruption of blood vessels and virus dissemination to neighboring cells, including cells in the brain (Ludwig et al., 2011). It was therefore used as a positive control to visualize BBB disruption.

To evaluate changes in the vascular endothelium, we inoculated fli1a:GAL4FF/5xUAS:RFP transgenic fish (hereafter referred to as fli:G/U:RFP), which express RFP in endothelial cells of the brain as well as of peripheral vessels. IHNV-infected fish were fixed at 1 and 2 dpi and immunolabeled using the 19B7 mAb directed against the G protein of IHNV. As opposed to control uninfected fish (Figure 3A), all stained fish showed disrupted vessels and scattered likely apoptotic RFP-positive cells (Figure 3B; Figure S3A; Movie S1.

Related to Figure 3). On the other hand, we monitored SINV-infected fli:G/U:RFP fish over a period of 7 days and overall, the vasculature looked intact, even in larvae showing an extensive infection in the CNS (Figure 3C;

Figure S3B; Movies S2 and S3. Related to Figure 3) and was comparable to control uninfected fish (Figure 3A).

To ensure optimal imaging of even deep brain vessels, SINV-infected fli:G/U:RFP larvae were processed using the recently developed Clarity protocol and later imaged at high resolution. Imaging revealed no apparent disruption of brain microvasculature (Figure 3D). Infected cells were sometimes adjacent to cells of the vasculature, especially in the periphery, but no co-localization was ever observed (for 15 animals examined in the brain, in 3 independent experiments, and for 13 animals examined in the periphery) (Figure S3C; Movie S4. Related to Figure 3).

In conclusion, our data show that SINV does not infect nor disrupt the BBB endothelium to gain access to the CNS (Figure 3 In addition, we depleted them specifically through metronidazole treatment of mpeg:G/U:Nfsb-mCherry larvae, exploiting the catalytic ability of the NfsB-mCherry fusion protein as previously described [START_REF] Davison | Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish[END_REF] (Figure S4A and S4B. Related to Figure 4).

Macrophage depleted SINV-infected larvae displayed a slight increase in disease severity -assessed using the scoring system previously developed (Palha et al., 2013) -(5.2 ± 1.1 N=24 compared to 3.6 ± 1.0 N=24 in controls) but no significant increase in mortality (Figure 4B). By 7 dpi, both controls and treated fish displayed a similar rate of infection (~50%) in the CNS (Figure 4C; Figure S4C. Related to Figure 4), although treated fish showed a bigger variability in terms of appearance of infected cells in the CNS.

We could therefore exclude entry of SINV in the CNS via infection of macrophages (Figure 4; Figure S4).

An eye model of infection shows that SINV can be transported via axons to the CNS after infection of peripheral neurons

Several viruses infect and replicate in peripheral nerves [START_REF] Koyuncu | Virus infections in the nervous system[END_REF].

These are connected to neurons of the CNS and act as springboard for efficient entry and replication in the brain. In the case of SINV infection, we sometimes observed infection of peripheral sensory neurons such as trigeminal ganglion cells prior to infection in the CNS. Early infection of muscle cells followed by appearance of GFP + cells among motor neurons at the corresponding level of the spinal cord was also a common occurrence (Figure 5A). These observations strongly suggest axonal spreading of the virus to the CNS.

To directly test if axonal transport of SINV can occur, we exploited the well-known connectivity of the visual system [START_REF] Grove | Turning neurons into a nervous system[END_REF]. We injected the virus in the retina, in an area comprised between the ganglion cell layer and the outer nuclear layer (Figure 1A). Fish injected in the eye did not show an increase in mortality rates and overall disease scores were comparable to those obtained in iv inoculated fish (data not shown). Injections in the dense retinal parenchyma is difficult and leakage of inoculum to other tissues probably occurred; however, leakage can be predicted to predominantly result in infection of areas close to the injected eye, while axonal transport should result in infection of the contralateral optic tectum (Figure 5B). All infected larvae displayed GFP in the retina at 1 dpi and 45% showed also infection of the contralateral optic tectum.

Altogether, because we excluded alternative possibilities, these data indicate that infection of peripheral nerves and subsequent axonal transport is the major route of CNS access for SINV (Figure 5).

SINV-infected larvae mount a strong IFN-mediated immune response

In all vertebrates, type I interferons (IFNs) represent the first line of defense and the main cytokines involved in protection against viral infections. The type I IFN response of larval zebrafish is substantially mediated by IFNφ1 and IFNφ3 (Aggad et al., 2009;Palha et al., 2013). qRT-PCR analyses of RNA extracted from SINV infected larvae revealed that the virus induces high mRNA levels of both ifnφ1 and ifnφ3 as well as of interferon stimulated genes (ISGs), such as rsad2 (also known as viperin or vig1). The peak of expression was observed at 1 dpi and was followed by a slow (ifnφ1, vig1) or relatively rapid (ifnφ3) decrease in mRNA levels over time (Figure 6A-C). Fluorescence microscopy of ifnφ1:mCherry transgenic larvae (Palha et al., 2013) revealed two main IFN producing cell types during SINV infection: hepatocytes and motile leukocytes.

Many mCherry-positive leukocytes were observed (Figure 6D) as early as 2 dpi and increased steadily over time (data not shown). To determine the relevance of the response, we knocked-down (kd) all type I IFN receptors (IFN-R) through injection of antisense Morpholino Oligonucleotides (MOs) directed against the CRFB1 and CRFB2 subunits (Aggad et al., 2009). The survival rate of IFN-R morphants dropped to about 50% after infection (Figure 6E) and the disease severity increased sharply, with respect to embryos injected with a control MO and later infected with SINV (Figure 6F). Surprisingly however, IFN-R morphants did not develop brain infection at earlier time-points or at higher frequency with respect to controls (Figure 6G). Nevertheless, we observed an extensive increase in the number of infected cells in IFN-R morphants (Figure 6H-I) and more cell types became susceptible to SINV (e.g. chondrocytes, fibroblasts -data not shown).

RNA interference (RNAi) is used by plants and many invertebrates as a major antiviral defense mechanism [START_REF] Kemp | Antiviral immunity in Drosophila[END_REF], and whether an antiviral function for RNAi exists in mammals has been a long-standing debate [START_REF] Umbach | The role of RNAi and microRNAs in animal virus replication and antiviral immunity[END_REF]. Thus, we tested whether zebrafish larvae show evidence of a RNAi mediated response against SINV, in parallel with the IFN response, by performing deep sequencing of small RNAs extracted from 1 dpi SINV-infected larvae. Unlike what happens in SINV-infected Drosophila (Saleh et al., 2009), no SINV-specific small RNAs produced through the RNAi machinery could be detected in zebrafish (data not shown). These results show that zebrafish larvae mount a protective IFN response against SINV, which limits infection in the periphery but is not sufficient to prevent neuroinvasion (Figure 6).

Discussion

Invasion of the CNS is a key event during viral infections, and often a matter of life and death for the host. It is however quite difficult to observe this phenomenon in mammals, thereby hindering the design of preventive strategies.

In the present study, we used zebrafish as a model to study the infection course of SINV, with a focus on its neuroinvasiveness. We found that the peak of viremia is at 48 hpi and it slowly declines over time. A similar kinetic was obtained for the IFN-mediated immune response. Observed clinical signs included edema, yolk opacification, and blood flow arrest. Thanks to in vivo imaging studies, we also assessed the progression of the infection. Infected cells appeared in the periphery as early as 8 hpi, always prior to infected neurons of the peripheral or the central nervous system. Overall, the disease signs and kinetic of SINV infection in zebrafish were quite similar to those previously observed in CHIKV-infected fish (Palha et al., 2013), suggesting that the mechanisms that determine disease onset are conserved between related Alphaviruses.

While ic injections resulted in rapid onset of CNS infection in 100% of cases, iv injected larvae displayed a greater variability in terms of time of appearance and progression of CNS infection. This phenomenon was also observed in inbred mice injected with SINV subcutaneously (Cook and Griffin, 2003), and therefore suggests that the variability observed is not due to the higher genetic heterogeneity of zebrafish, which are not fully inbred. However, iv injections better represent the actual entry route of the virus upon bite from an infected mosquito and therefore allowed us to characterize the natural mechanism of SINV entry in the CNS.

Fleming et al. showed that the maturation of the BBB in zebrafish larvae is progressive until 10 dpf. At 3 dpf, the time at which larvae were injected, high molecular weight molecules (~900 Da) are already excluded from the CNS (Fleming et al., 2013). We could therefore exclude passive transport of viral particles from the blood. In some cases, viruses have been shown to enter via the choroid plexus (ChP) (e.g. HIV, Falangola et al., 1995), the region of the brain where the cerebrospinal fluid is formed. Studies on morphogenesis formation have shown that in zebrafish the ChP develops at the dorsal midline on the fourth ventricle and shifts towards the ear level at around 3 dpf (García- [START_REF] García-Lecea | In vivo analysis of choroid plexus morphogenesis in zebrafish[END_REF]. Here we found that commonly infected areas in the brain were rather lateral or distant from the ChP (e.g. olfactory bulb, optic tectum, medulla), which strongly suggests that SINV does not enter the brain via prior infection of the forming ChP.

The vasculature was shown to remain intact and infection of endothelial cells of the brain microvessels was never observed. Moreover, ic injections revealed little or no infection of cells outside the CNS, thereby corroborating the observation that SINV does not compromise the integrity of the BBB.

We also showed that the virus does not target macrophages. Macrophage depletion assays induced a slightly higher disease severity in treated fish, as in the CHIKV model, but did not prevent the virus from accessing the CNS. These results show that, even though macrophages may contribute in part to the control of SINV infection, they are not required for the virus to reach the CNS.

The observations we collected from SINV-inoculated larvae therefore strongly support the hypothesis that viral entry in the CNS occurs after infection of peripheral nerves, such as infection of trigeminal ganglion cells prior to infection in the CNS or infection of muscle cells followed by infection of motor neurons.

These results are in accordance with the aforementioned study of mice infected with a luciferase-recombinant SINV strain, where viral replication in the nose or the spinal cord was shown to always precede infection in the brain (Cook and Griffin, 2003).

As previously observed in CHIKV-infected larvae, IFN was highly induced with the expression peaking at 24 hpi and slowly declining over time. Moreover, we showed that IFN-R kd induces changes in viral tropism and determines infection of several cell types not normally targeted by the virus in zebrafish. This is in accordance with a previous study conducted in mice by Ryman et al. (2000).

Interestingly, while it did result in more infected cells, IFN-R kd did not cause an increase in occurrence of CNS invasion. Overall these data indicate that the IFN-mediated immune response is a major component involved in the protection against SINV infection and that IFNs limit SINV replication efficiency at early stages post-infection in the periphery, yet they do not prevent entry in the CNS.

Other more complex immune mechanisms are probably involved in the response against SINV once the virus reaches the CNS, which are worth investigating in the future.

While IFN was clearly a major defense mechanism of zebrafish larvae against SINV, no evidence for a RNAi-mediated response could be detected. Our simple experiment does not rule out the existence of antiviral RNAi in fish. However, since it has been proposed that the RNAi response is restricted to undifferentiated stem cells in mammals, and therefore more prominent in the early life stages [START_REF] Maillard | Antiviral RNA interference in mammalian cells[END_REF], our use of zebrafish larvae reinforces our conclusions.

Moreover, a recent study provided compelling evidence for the absence of an antiviral role of RNAi in mammals (Backes et al., 2014). Since antiviral RNAi exists in plants, insects, and nematodes, it is very likely to be ancestral, and thus would have been lost in the lineage leading to mammals. If, as we believe, teleost fish do not use antiviral RNAi, then this loss occurred more than 350 My agovery possibly when the protein-based antiviral defense appeared in early vertebrates.

Understanding the entry mechanism of a certain pathogen in the CNS has always been impaired by the difficulties in visualizing the progression of the infection.

Moreover, a high number of animals and organ/tissue samples are generally needed at different time-points, to avoid missing important cues. The combined use of a recombinant SINV-GFP strain with fluorescent-reporter zebrafish lines, on the contrary, allowed us to gain single-cell resolution details at the whole organism level and with minimal invasiveness for the host. This in turn will be useful in the future for the study of neurotropic viruses and to test new antiviral therapeutics in vivo.

Experimental Procedures

Ethical statement

Animal

Fish lines and husbandry

Zebrafish embryos were raised according to standard procedures as previously described [START_REF] Westerfield | The Zebrafish Book: A guide for the laboratory use of zebrafish (Danio rerio)[END_REF][START_REF] Levraud | In vivo analysis of zebrafish innate immunity[END_REF]. Wild-type AB zebrafish were initially obtained from ZIRC (Eugene, OR). The following transgenic and mutant lines were also used: Tg(elavl3:Gal4) zf349 [START_REF] Akerboom | Optimization of a GCaMP calcium indicator for neural activity imaging[END_REF], Tg(fli1a:Gal4FF) ubs4 [START_REF] Zygmunt | Semaphorin-PlexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1[END_REF] (Palha et al., 2013). Because of silencing issues of UAS-driven transgenes -a rare occurrence with the UAS:RFP nkuasrfp1a allelecareful selection of fish expressing the complete brain and vasculature pattern was performed at 3 dpf before inoculation.

Eggs obtained by natural spawning were bleached and raised at 28°C in Volvic source water. Eggs used for imaging purposes were raised in 1-phenyl-2-thiourea/Volvic (Sigma-Aldrich, 0.003% final) from 24 hours post-fertilization (hpf) onwards to prevent melanin pigment formation. At 3 dpf, just before infections, larvae that had not hatched spontaneously were manually dechorionated.

Viruses

SINV-GFP was produced on BHK cells, according to (Hardwick and Levine, 2000). The virus used corresponds to the pTE3'2J-eGFP strain (NC_001547.1), and was obtained by insertion of the eGFP gene under the control of a second subgenomic promoter (Hahn et al., 1992). Heat adapted IHNV strain 25.70 was produced on EPC cells as described previously (Ludwig et al., 2011).

Infection and disease scores

Injections and handling of larvae were performed as described [START_REF] Levraud | In vivo analysis of zebrafish innate immunity[END_REF]. Briefly, zebrafish larvae aged 70-72 hpf were inoculated by microinjection of ~10 2 TCID 50 viral SINV particles (~1 nL of supernatant from infected BHK cells, diluted to 10 8 TCID 50 /mL). Injections were performed either in the caudal vein/aorta or in the left optic tectum or in the left retina. Larvae were then distributed in individual wells of 24-well culture plates with 1ml water, kept at 28°C and regularly inspected with a stereomicroscope until 7 dpi. Clinical signs of infection were assessed first on aware animals, which were then anesthetized for better observation. Quantitative assessment of the clinical status was based on a precise list of criteria, as previously described (Palha et al., 2013). Briefly, clinical signs were assessed blindly, yielding a disease score ranging from 0 (no disease sign) to 15 (dead or terminally ill). For ethical reasons, all larvae used in the experiments were killed by anesthesia overdose at 7 dpi. IHNV infections were performed as described in (Ludwig et al., 2011). Briefly, larvae were injected iv with 10 2 PFU of IHNV25.70, distributed in individual 24-well plates and incubated at 24°C.

qRT-PCR

RNA extraction, cDNA synthesis and quantitative PCR were performed as previously described (Ludwig et al., 2011); externally quantified standards were included to provide absolute transcript amounts. The following pairs of primers (sense and antisense) were used: ef1a: GCTGATCGTTGGAGTCAACA and ACAGACTTGACCTCAGTGGT; SINV E1: GACAACATGCAATGCAGAATG and CTAGTCAGCATCATGCTGCA; ifnφ1 (secreted isoform):

TGAGAACTCAAATGTGGACCT and GTCCTCCACCTTTGACTTGT; ifnφ3:

GAGGATCAGGTTACTGGTGT and GTTCATGATGCATGTGCTGTA; rsad2:

GCTGAAAGAAGCAGGAATGG and AAACACTGGAAGACCTTCCAA. To normalize cDNA amounts, we used the housekeeping gene ef1a transcripts.

Time-lapse in vivo imaging

For in vivo time-lapse imaging, 5-10 larvae were anaesthetized with 112 mg/mL tricaine and immobilized in ~1% low melting-point agarose in the center of a 35-mm glass bottom Ibidi® dish, then covered with ~2 mL water containing tricaine. Transmitted light/fluorescence imaging was performed using a Leica SPE inverted confocal microscope equipped with a 10X (NA 0.30) dry objective and 16X (NA 0.5), 40X (NA 1.15), 63X (NA 1.30) oil immersion objectives. Imaging was typically performed at 26°C and Z-stacks with 2 µm steps were taken at least every 10 min. Fish were imaged every day beginning from 1 dpi up to 4-5 dpi with imaging sessions typically lasting 30 min; control uninfected larvae were always included.

Whole-mount immunohistochemistry (WIHC) and imaging of fixed samples

WIHC was performed as described [START_REF] Svoboda | Activity regulates programmed cell death of zebrafish Rohon-Beard neurons[END_REF]. The following primary antibodies were used: chicken polyclonal to GFP (1:500, Abcam), mouse mAb to acetylated tubulin (1:1000, Sigma), rabbit polyclonal to DsRed (1:500, Clontech), which also labels the mCherry protein, 19B7 mouse mAb antibody specific against IHNV G protein (glycoprotein) (1:500) [START_REF] Biacchesi | Heterologous exchanges of the glycoprotein and the matrix protein in a Novirhabdovirus[END_REF]

Imaging in high refractive index solution

A fructose based high refractive index solution (fbHRI) was prepared as follows:

70% fructose (wt/vol), 20% DMSO (wt/vol) in 0.002 M PBS, 0.005% sodium azide (wt/vol). The refractive index of the solution was adjusted to 1.4571 using a refractometer (Kruss). In preparation for imaging, the samples were incubated in 50% (vol/vol) fbHRI for 6 hours and finally incubated in fbHRI for at least 12 hours. For imaging, samples were mounted in 1% (wt/vol) low melting-point agarose and covered with fbHRI. Whole-mount larvae fluorescence was recorded with a Leica TCS SP8 two photon microscope equipped with a mode locked Ti:Sapphire laser (Chameleon, Coherent) at 770 nm and the Leica HC FLUOTAR L 25x/1.00 IMM motCorr objective.

Macrophage depletion

Metronidazole-mediated depletion was performed as described in [START_REF] Davison | Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish[END_REF]. Briefly, Tg(mpeg:Gal4FF) gl25 fish [START_REF] Ellett | Mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish[END_REF] were crossed to Tg(UAS-E1b:Eco.NfsB-mCherry) c26 [START_REF] Davison | Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish[END_REF], to generate double-positive transgenics and single-positive sibling controls. Embryos were placed from 48 hpf to 72 hpf in a 10 mM Metronidazole/0.1% DMSO solution to induce specific depletion of NfsB-mCherry-expressing macrophages. Embryos were then rinsed 3X with embryo water and subsequently injected with the virus.

Morpholino injection

Morpholino antisense oligonucleotides (Gene Tools) were injected into 1-2-cell stage embryos as previously described [START_REF] Levraud | In vivo analysis of zebrafish innate immunity[END_REF]. crfb1 splice morpholino (CGCCAAGATCATACCTGTAAAGTAA) (2 ng) was injected together with crfb2 splice morpholino (CTATGAATCCTCACCTAGGGTAAAC) (2 ng), knocking down all type I IFN receptors (Aggad et al., 2009). As control a morpholino with no target (NRP) was used: (GAAAGCATGGCATCTGGATCATCGA) (4 ng).

Statistical analysis

To evaluate difference between means, a two-tailed unpaired t-test or an analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test were used, when appropriate. Normal distributions were analyzed with the Kolmogorov-Smirnov test. Non-Gaussian data were analyzed with a Kruskal-Wallis test followed by Dunn's multiple comparison test. P<0.05 was considered statistically significant (symbols: ***P<0.001; **P<0.01; *P<0.05; ns=not-significant). Survival data were plotted using the Kaplan-Meier estimator and log-rank tests were performed to assess differences between groups.

Statistical analyses were performed using Prism 5 software.

CHAPTER II. CNS-Specific Immune Responses to SINV infection

The results presented in the previous Chapter relative to the IFN-mediated immune response upon SINV infection have shown that 1. the IFN response, measured at the whole-body level, is readily activated and peaks at 24 hpi, which coincides with the peak of viremia, 2. the main IFN-producing cell populations are hepatocytes and motile leukocytes, most likely neutrophils, and 3. IFN knock-down results in changes in virus tropism, increased disease severity and mortality but no significant increase in brain infection frequency. In accordance with the results from Ryman et al., we have shown that the IFN response is crucial to contain virus spreading in the periphery at early times post-infection (Ryman et al., 2000). Our data also indicated that IFN production is not sufficient to prevent CNS invasion, and even less efficient to stop the progression of the infection in the CNS than in the periphery. Based on these results, we started a more detailed investigation of the immune responses activated in the CNS. In particular, the study of different SINV strains has given us insights into strain-specific modulations of host responses that determine from early time points the outcome of the infection (section 2.1). On the other hand, the further characterization of the ifnφ1:mCherry line has helped us shed light into the events occurring at later time points, after CNS invasion (section 2.2). Although preliminary, these data are nonetheless very promising and will continue to be explored in the future.

Strain-dependent modulation of the IFN signaling pathway

The outcome of an infection heavily depends upon a number of factors, including the genotype of the pathogen as well as the age and immune competence of the host, and is usually decided at the early stages. Rapid activation of the innate antiviral immune response is in fact crucial to block permissivity of uninfected cells to infection. In turn, subversion of the IFN response results in quick and generalized virus spread, even in cells not normally targeted by the virus. The characterization of different virus strains can therefore be helpful to determine conserved virulence determinants between members of the same virus family as well as their specific interaction with the immune system.

This was one of the reasons that lead us to produce a different SINV recombinant strain, SINV pTR339-mCherry/2A (obtained from the original African isolate AR339 and from hereon referred to as SINV2-mCherry), to compare its infectious course with that of our first SINV strain, pTE3'2J-eGFP (which we will now rename SINV1-GFP) (Hahn et al., 1992). The other obvious reason was the color complementarity between the two strains.

SINV AR339 was originally isolated from a pool of Culex pipiens and Culex univittatus mosquitoes collected in Egypt [START_REF] Taylor | Sindbis virus: a newly recognized arthropod-transmitted virus[END_REF]. From that strain, a full-length cDNA infectious clone (TR339) was generated [START_REF] Mcknight | Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes[END_REF]. In contrast, the recombinant SINV strain TE3'2J was constructed from a chimeric mouse neurovirulent variant of AR339 called TE12 (Lustig et al., 1988). TCID 50 /mL) revealed that the strain was much more virulent than SINV1-GFP.

Comparison of the two strains in terms of viral burden and IFN induction at 1 dpi showed that SINV2-mCherry replicates at higher levels (~6 times) while IFN expression was about 2 times lower than that measured in SINV1-GFP infected larvae. Moreover, SINV2-mCherry kills over 60% of injected larvae as opposed to SINV1-GFP, which instead shows ~20% mortality rate at 7 dpi, with a similar inoculum (Figure R2.2). Imaging of fixed samples revealed that SINV1-GFP targets a restricted number of cell types as opposed to SINV2-mCherry, whose infection resembles that observed in IFN-R knock-down morphants (Figure R2.3).

Nevertheless, both strains showed infection of neuronal cell types, such as trigeminal ganglion cells (Figure R2.4).

In the case of SINV2-mCherry, however, infection in the CNS was observed in all injected larvae while SINV1-GFP reached the brain in only 40% of cases. To compare the two strains in terms of cell tropism, we performed injections of serial dilutions of SINV2-mCherry and found that 10 6 TCID 50 /mL was a concentration phenotypically comparable to that of SINV1-GFP. Finally, co-injection of SINV1-GFP (10 8 TCID 50 /mL) and SINV2-mCherry ( 10

Leukocyte recruitment in the CNS

All experiments described in this and the following sections have only been performed with SINV1-GFP (SINV).

To better characterize the role of IFN-producing cells in the control of SINV-induced CNS infection, we injected ifnφ1:mCherry transgenic larvae and performed in vivo imaging at daily intervals as previously described. Unlike what was previously reported in CHIKV infection in zebrafish (Palha et al., 2013), IFN-producing motile cells (80% of which were neutrophils in the CHIKV system) were not excluded from the CNS (from which neutrophils are excluded in healthy animals) but rather actively permeating into the brain, even when the infection was relatively contained to one or few infection foci (Figure R2.6).

The morphology and motility of these cells indicated that they were most likely neutrophils; this has been formally verified by injecting SINV in LyZC:DsRed transgenic larvae (a red neutrophil reporter line) and observing the same response reported for ifnφ1:mCherry transgenic larvae.

IFN-expressing leukocytes were visible in the CNS already at 2 dpi, time at which most larvae begin to show CNS infection, and their number increased over time and later reached a plateau. Moreover, in the case of extended infection, they were shown to "uptake" SINV-infected cells in the brain (Movie R2.1; Movie R2.2;

Movie R2.3). Remarkably, within some infection foci, infected neurons were also expressing the IFN reporter. We had not observed that in our previous in vivo experiments, probably due to the lower laser power used to avoid phototoxicity (Figure R2.7). The quantification has shown that within 12 hrs the number of neutrophils close to a highly infected area does not change significantly. However, the number of infected neuronal cells decreases considerably. Additional analyses are needed to relate the number of events in which a neutrophil interacts with an infected cell to that in which an infected cell disappears, thereby proving that neutrophils are needed to clear the infection in the CNS. Despite being incomplete for our purposes, the algorithm shows great potentials and helped reducing considerably the time required to perform such an analysis manually.

CHAPTER III. SINV Spreading and Persistence Mechanisms

Upon injection of SINV into zebrafish larvae, we showed that the virus enters the CNS most likely through infection of peripheral neurons and subsequent retrograde axonal transport (Chapter 1 of the Results). In a few larvae infected intracerebrally, infection of spinal cord neurons and muscle cells following brain infection was also observed. This strongly suggests that the virus can also use the anterograde transport machinery and reach areas distant from the first infection foci. Spreading within the CNS occurred mostly via long distance axonal transport, as evidenced by infection of Mauthner neurons. These are bilaterally symmetric reticulospinal neurons whose axonal projections are distributed from the caudal midbrain to the spinal cord [START_REF] Zottoli | The Mauthner cell: what has it taught us?[END_REF].

Unlike what occurs in CHIKV-infected zebrafish larvae (Palha et al., 2013), SINV-infected cells in the CNS did not all survive very long. In accordance with the results obtained in mice from Lewis et al. (1996), apoptotic cells were observed in several infected areas and correlated with the increasing number of circulating leukocytes in the brain (Movie R3.1).

To better document viral persistence over neuronal cell death in the CNS, we started setting up a feeding protocol for the long-term maintenance of infected larvae. Larvae can begin to feed between 4-5 dpf, time at which they exit the chorion and the mouth begins to open. Larvae raised in the fish facility are normally kept in groups of 50 or 60 in 2 liter tanks, with 3 rounds of food per day and regular water changes. However, to monitor the progression of the infection in our injected larvae, we are obliged to keep them in separate wells and feed them regularly while avoiding contamination of the water from residual food.

Adapting a method previously developed for the feeding of individual axenic larvae [START_REF] Rendueles | A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria[END_REF], we fed larvae daily with Tetrahymena thermophila and followed the progression of SINV infection in the CNS by daily observation at the fluorescent stereomicroscope. In a preliminary experiment, the majority of iv infected larvae succumbed within 4 to 5 dpi and only 20% survive until 21 dpi, end point of the experiment. However, only 8% of total infected larvae also retained infection in the CNS for the whole duration of the experiment.

These experiments are so far inconclusive because the low surviving rates are inconsistent with the results normally obtained with unfed larvae in short-term experiments (within 7 dpi). In all likelihood, there was a problem with the feeding procedure, which will need to be solved before a new test. Nevertheless, they showed that, in some cases, SINV infection can persist for at least three weeks in the CNS.

With the aim to characterize the effect of SINV on survival and response of neuronal cells, we also established a protocol to obtain primary cultures of zebrafish brain cells. This consists in the dissection of 72 hpf zebrafish larvae, to isolate brain samples before enzymatic and mechanical dissociation. We hope that this newly developed tool will be helpful to identify the transport mechanism employed by SINV in neuronal cells, SINV effect on cell survival, role of autophagy, and cell responsiveness to IFN before and after the infection.

CHAPTER IV. Tests on a Potential Broad-Spectrum Antiviral Drug

In the context of a collaboration with the "Viral populations and Pathogenesis"

Unit headed by Marco Vignuzzi at Pasteur Institute, we also recently started investigating the potential antiviral activity of Difluoromethylornithine (DFMO) against SINV infection in vivo.

DFMO is a drug that irreversibly inhibits ornithine decarboxylase 1 (ODC1), a critical enzyme in polyamine biosynthesis [START_REF] Metcalf | Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E.C.4.1.1.17) by substrate and product analogs[END_REF]. These are small positively-charged molecules derived from arginine, involved in several cellular processes, including cellular proliferation [START_REF] Gerner | Polyamines and cancer: old molecules, new understanding[END_REF], apoptosis [START_REF] Schipper | Involvement of polyamines in apoptosis. Facts and controversies: Effectors or protectors? Semin[END_REF], ion channel regulation [START_REF] Williams | Interactions of polyamines with ion channels[END_REF],

DNA conformation [START_REF] Thomas | Polyamine-mediated conformational perturbations in DNA alter the binding of estrogen receptor to poly(dG-m5dC).poly(dG-m5dC) and a plasmid containing the estrogen response element[END_REF], transcription [START_REF] Frugier | Synthetic polyamines stimulate in vitro transcription by T7 RNA polymerase[END_REF][START_REF] Law | Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase[END_REF], and translation [START_REF] Mandal | Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells[END_REF] in both mammalian and non-mammalian cells; their exact mechanism of action, however, is generally unclear. In the case of Semliki Forest virus and other RNA viruses, they have also been shown to promote viral RNA synthesis [START_REF] Tuomi | Synthesis of Semliki-forest virus in polyamine-depleted baby-hamster kidney cells[END_REF]. Therefore, we placed 24 hpf zebrafish embryos in 1 mM DFMO-containing water for three days, and later measured polyamine content from fish homogenates with thin layer chromatography. In this case, the embryos developed normally, and underwent a significant reduction in polyamines quantity, thereby showing that DFMO is active in larvae as well (Figure R4.1A). Three days after treatment, zebrafish were inoculated ic with ∼10 2 TCID50 SINV as previously described.

Viral titers enumerated at 1 and 2 dpi were significantly reduced in DFMO-treated zebrafish (Figure R4.1B), showing the potential in vivo antiviral activity of DFMO.

This was corroborated by tests in other species, and with other viruses, confirming the potential of DFMO as a broad-range inhibitor of RNA virus replication that could be useful during viral outbreaks. This work is currently being submitted for publication (Mounce et al., submitted).

Future experiments will focus on the determination of the exact mechanism of action of DFMO in the context of suppression/reduction of viral replication in zebrafish infected larvae.

DISCUSSION

The major results obtained during this PhD work have led to the characterization of the entry mechanism of Sindbis virus (SINV) into the Central Nervous System (CNS) of zebrafish larvae. We have shown that the virus neither disrupts the Blood-Brain Barrier (BBB) nor infects macrophages to reach the CNS. Instead, our data is consistent with axonal transport to the CNS after infection of peripheral neurons or nerve termini.

In the submitted article, we have identified neurons as an important target of the virus, thanks to in vivo analyses of elavl3:Gal4/5xUAS:RFP infected larvae (HuC:G/U:RFP) and ex vivo after co-immunolabeling of infected cells and axonal projections. However, we have not been able so far to determine the precise composition of the cell subtypes infected in the CNS. Infection of our glia-specific transgenic line (Gfap:eGFP) [START_REF] Bernardos | GFAP transgenic zebrafish[END_REF] was not possible as it is a GFP-expressing transgenic line, and at the time our SINV strain was only available as a GFP recombinant. Moreover, we have tested several antibodies directed against glial cells (anti-GFAP, anti-Glutamine Synthetase) but have failed to observe a specific staining in the CNS. This could be due to the fact that this cell type is not mature at the time of the infection and therefore does not express a yet usable marker (the same antibodies give a reproducible staining in adult zebrafish). We have encountered the same difficulties when trying to assess the percentage of post-mitotic over undifferentiated neurons, as our antibody directed against neurogenin (a marker of undifferentiated neurons) also resulted in non-specific signal. This represents a drawback for the use of zebrafish with respect to other model organisms, as fewer biochemistry tools have been set up to date. We hope, nevertheless, that the use of the Clarity protocol together with the neuroanatomy expertise provided by the group of Jean-Stéphane Joly in Gif-sur-Yvette will help us better characterize the specific neuronal subtypes targeted by the virus. In addition, we now have the isogenic non-GFP pTE3'2J strain, which can be used in GFP transgenic larvae; the infection cannot be followed live but, in fixed larvae, infected cells can be detected with our anti-capsid antibody.

At the same time, we have focused our attention on the IFN-mediated immune response both at the whole-organism as well as at the CNS-specific level and have Despite some initial similarities with respect to the kinetic of replication and of the immune response, we observed many differences when comparing the infection course of SINV and Chikungunya virus (CHIKV) in zebrafish larvae.

Unlike CHIKV, SINV exhibited a stronger neurotropism and induced recruitment of IFN-producing cells in the CNS upon infection (section 3 of the Discussion).

However, the infection course described for SINV with respect to its entry mechanism in the CNS, its neurotropism, and the variability observed in different animals (section 4 of the Discussion) is in accordance with several important studies conducted in mice.

Identification of Viral Factors of Neuroinvasion

The results obtained from the study of different SINV variants in Chapter 2 section 2.1 of the Results have shown that the two strains differ from each other in terms of:

-Viral burden, with SINV2-mCherry replicating at higher levels infected neurons) when expressing a palGFP or a palDsRed fluorescent reporter [START_REF] Furuta | In vivo transduction of central neurons using recombinant Sindbis virus: Golgi-like labeling of dendrites and axons with membrane-targeted fluorescent proteins[END_REF]. Therefore, in order to make certain that the observed differences are not linked to the insertion of the fluorescent protein, we will compare the infection of SINV1-GFP with that of SINV2-GFP. These experiments will be conducted soon, as SINV2-GFP has already been produced but requires a further concentration step. However, the two insertions encode proteins of comparable size with no plausible relevance to virulence. It seems quite unlikely that they would result in such significant differences; hence, we will assume they do not for the rest of the discussion.

-
The differences observed between these two strains in terms of viral burden in zebrafish correlate with those observed in mosquitoes. Examination of SINV TR339 propagation (from which SINV2-mCherry was derived) in Aedes aegypti mosquitoes showed a greater than 90% midgut infection rate at 7 dpi [START_REF] Myles | Comparison of the transmission potential of two genetically distinct Sindbis viruses after oral infection of Aedes aegypti (Diptera: Culicidae)[END_REF]. In contrast, SINV TE3'2J (SINV1-GFP progenitor) was shown to infect less than 15% of mosquitoes when analysed similarly [START_REF] Pierro | Development of an orally infectious Sindbis virus transducing system that efficiently disseminates and expresses green fluorescent protein in Aedes aegypti[END_REF].

This may be purely stochastic, since the virus can be expected to rely on distinct sets of host cellular components for its replication in different species. However, it is interesting to note that a common feature of the zebrafish and mosquito infections is their lower body temperature as compared to mammals. SINV shuttles between insects and birds and must be able to replicate from about 20 to 42°C (68 to 107°F). Each strain probably has an optimal temperature and this suggest it may be closer to 28°C (82°F) for SINV2-mCherry than for SINV1-GFP.

Moreover, the higher viral burden measured in SINV2-mCherry injected larvae could be related to its broadened cell tropism. In turn, this can be associated with the lower IFN induction observed. SINV2-mCherry may induce host transcription and translation shutoff in infected cells, thereby blocking IFN and/or ISG expression. The disease signs and tropism observed in SINV2-mCherry infected larvae were indeed reminiscent of those observed in IFN-receptor (IFN-R) morphants injected with SINV1-GFP. Thus far we have not tested in vivo whether IFN producing cells are indeed infected with SINV2-mCherry and if this, in turn, results in decreased levels of IFN expression. However, the ability to modulate IFN expression has been reported also for several other SINV strains and recombinant mutants. Cruz et al. showed that mutations at the P3 cleavage site of SINV AR86 (another SINV isolates) determine a higher induction of IFN with respect to the wild type isolates [START_REF] Cruz | Modulation of type I IFN induction by a virulence determinant within the alphavirus nsP1 protein[END_REF], although this modulation was independent from virus mediated host shutoff. Frolov et al. showed the importance of IFN induction early after infection (2-4 hpi), to block virus spreading and indicated mutations in nsP2 as the factors involved in host defense induction [START_REF] Frolov | Early events in alphavirus replication determine the outcome of infection[END_REF]. Indeed, one mutation resulting in an amino acid change in the NSP2 protein (Pro 978 Leu) is present in SINV2-mCherry as compared to SINV1-GFP.

Interestingly, the higher neuroinvasiveness observed in SINV2-mCherry-infected larvae did not correlate with that measured in SINV1-GFP injected IFN-R morphants. This difference therefore must be linked to specific SINV2-mCherry determinants that are independent from its ability to modulate IFN. These determinants may be absent in SINV1-GFP, thereby explaining its unchanged neuroinvasiveness in IFN-R morphants. SINV neurovirulence has been directly linked to specific amino acid changes in the E1 and E2 genes and mutations present in either of the two can determine differences in the observed neurovirulence (Lustig et al., 1988;[START_REF] Tucker | Mechanism of altered Sindbis virus neurovirulence associated with a single-amino-acid change in the E2 Glycoprotein[END_REF]. As a consequence, strains with specific E1 and E2 mutations have been shown to induce significant levels of apoptosis in brains and spinal cord of weanling but also of older mice (Lewis et al., 1996). Comparison of the genomic cDNA sequences of SINV1-GFP and SINV2-mCherry identified two amino acid changes in E2 (Arg 338 Cys; His 383 Gln). In the future we will try to isolate the specific residues that determine SINV2-mCherry neuroinvasiveness by building various SINV1/SINV2 chimeras, and using in vivo imaging of infected zebrafish larvae as readout for the neuroinvasive phenotype.

Leukocyte Behavior in Response to SINV Infection in the CNS

The data acquired on the cellular immune response against SINV infection suggest that neutrophils represent the main IFN producing cells, in accordance with the results obtained by Palha et al. in CHIKV infected larvae (Palha et al., 2013).

However, they were not excluded from the brain but rather accessing it rapidly, sometimes also in cases in which infection in the CNS was relatively contained.

Moreover, they were observed directly interacting with infected cells in the brain.

These were unexpected findings since neutrophils, which are strictly excluded from the brain of healthy vertebrates, had not been observed entering the brain of CHIKV-infected zebrafish larvae, despite persistent CNS infection (Palha et al., 2013). Nevertheless, this "neutrophil invasion" phenotype may be quite relevant for the study of viral encephalitis, since neutrophils have been shown to invade the CNS during encephalitis caused by other viruses, such as Herpesviruses [START_REF] Silverman | Persistent cerebrospinal fluid neutrophilia in delayed-onset neonatal encephalitis caused by Herpes simplex virus type 2[END_REF] or West Nile virus [START_REF] Davis | West Nile virus neuroinvasive disease[END_REF].

Future experiments will be directed towards the understanding of the exact role played by neutrophils during CNS invasion. In particular, we will deplete them specifically using the metronidazole-mediated depletion system [START_REF] Davison | Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish[END_REF], to determine their importance in the control/eradication of SINV infected neuronal cell types. It may also be worth testing for possible deleterious side effects of the presence of neutrophils in the CNS, for instance using behavioral testing, some of which exist for zebrafish larvae [START_REF] Lange | Inter-individual and inter-strain variations in zebrafish locomotor ontogeny[END_REF], although this would require a setup compatible with the use of infected larvae.

Another aspect that needs to be further addressed is the role of microglia in the control of SINV infection in the CNS. Our preliminary data suggest that microglia are not capable of restricting the infection in the CNS. Very few cells were observed close to infection sites and, according to their rounded shape, they were possibly undergoing apoptosis. These experiments were performed ex vivo on fixed larvae; therefore, a more detailed analysis in vivo is necessary. Nevertheless, these observations are in accordance with the results obtained after macrophage depletion in the mpeg:G/U:Nfsb-mCherry larvae. We showed in fact that their absence did not result in increased mortality rates, thereby suggesting that this cell population plays a minor role, if any, in controlling SINV infection.

Finally, we will perform comparative in vivo analyses on the expression patterns of ifnφ1 and ifnφ3, thanks to the newly generated ifnφ3:GAL4FF line. The transgene was generated in the laboratory of George Lutfalla in Montpellier and the line was produced by the AMAGEN transgenesis platform at Gif-sur-Yvette.

The line will be crossed to UAS:GFP or UAS:RFP fish, and the resulting double-transgenic larvae will serve the purpose of characterizing IFNφ3 producing cells in the context of both CHIKV and SINV infection. At the transcriptional level in fact we measured high expression levels of ifnφ3, which peaked at 24 hpi (like ifnφ1) and then quickly decreased (unlike ifnφ1). This new transgenic line will be useful to analyse cell population or tissue specific expression of IFNφ1 and IFNφ3. Our preliminary results indicate that IFNφ1 and IFNφ3 are expressed by non-overlapping cell populations. In the case of IFNφ1,

we have been able to observe its expression not only in leukocytes and infected hepatocytes, but also in infected neurons. However, a more detailed quantification is required to determine the relative contributions of these expression patterns. At the same time, we will follow IFN-responding cells with the help of the MxA:mCherry line, also developed by George Lutfalla.

SINV and CHIKV Infection in Zebrafish

The characterization of the infection course of CHIKV in zebrafish larvae provided two important findings: 1) Several cell types are targeted by the virus, including cells of the brain parenchyma, but only brain cells remain infected for several days. 2) A potent IFN-mediated response is induced. IFN is mostly expressed by neutrophils, though these are excluded from the CNS, and knockdown of IFN receptors results in higher viral loads and dramatic increase in lethality following CHIKV infection (Palha et al., 2013). However, due to its classification as a BSL3 pathogen, CHIKV could only be studied ex vivo on fixed samples or in vivo but under very restrictive conditions (BSL2 + facility, low-resolution microscopy). Therefore, in order to better characterize the viral infection course in the CNS and the relative immune response but with less security restrictions, we decided to employ the closely related SINV as a model virus with neurotropic activity. The initial characterization of its mortality rate, using the SINV1-GFP strain, and kinetic of infection gave comparable results with respect to those observed in CHIKV-infected larvae. Both viruses replicate efficiently, with replication peaking at 24 hpi, and show relatively low mortality rates (10% for CHIKV and 20% for SINV). The kinetic of the immune response is also comparable between CHIKV and SINV, and follows the kinetic of virus replication. However, while CHIKV targets predominantly muscle cells, fibroblasts, hepatocytes and endothelial cells, SINV rarely infects fibroblasts, it has never been observed in co-localization with cells of the vascular endothelium, and is predominantly neurotropic. Moreover, CHIKV infection of the CNS occurs in almost all infected larvae, but is self-limited to a few cells, while SINV reaches the CNS in ~50% of infected larvae, where it expands. These differences suggest that the two viruses may employ different mechanisms to enter the CNS. While CHIKV could possibly infect endothelial cells of the brain microvasculature, SINV is instead transported via axonal projections of peripheral neurons.

However, a detailed characterization of CHIKV mode of entry is required in order to confirm this hypothesis. At the moment, CHIKV mechanism of neuroinfection remains largely ill-characterized, although it has been suggested to occur upon dissemination from the choroid plexus and the meninges in infected mice [START_REF] Couderc | A mouse model for Chikungunya: young age and inefficient type-I interferon signaling are risk factors for severe disease[END_REF]. It would be interesting to test this hypothesis in zebrafish larvae, although it will have to be performed ex vivo.

Another important difference between the two viruses lies in their effect on infected neuronal cells. While CHIKV-infected neurons have been shown to survive longer than other infected cell types (suggesting neurons could constitute a previously unknown CHIKV reservoir), SINV induces cell death in infected neurons (likely apoptosis), a phenomenon that was especially obvious in larvae with an extensive CNS infection. This in turn could be related with the observed infiltration of neutrophils in the brain upon SINV infection.

An important point that needs to be further characterized is the effect of IFN-R knock-down in SINV-infected larvae. Over 90% of CHIKV-injected IFN-R morphants succumb from the infection, demonstrating the protective role of IFN.

In the case of SINV-injected morphants, on the other hand, only 50% of them die after infection and the number of larvae presenting CNS infection does not increase with respect to control morphants. This is in contrast with the observation that infected neurons produce IFN and require IFN-producing cells to contain the infection in the CNS, which suggests IFN plays a more prominent role than the one observed in IFN-R morphants. However, since morpholino mediated knock-down is transient, it is very possible that its effect can only be felt on peripheral infection, which occurs first, and has worn off by the time IFN is induced in the CNS. This should be re-evaluated when true IFN or IFN-R mutants, obtained using CRISPR or other technique, will have become available.

Taken together, these observations suggest that SINV may be more closely related to other markedly neurotropic Alphaviruses than to CHIKV, and therefore a more detailed comparative analysis between the two is needed to identify conserved mechanisms of infection.

SINV Infection in Mice and Zebrafish

Despite its low pathogenicity in humans, SINV has been studied over several years in the mouse model to characterize virus-induced encephalitis. In particular, it was shown that SINV-induced lethality is dependent on its neurotropism as well as on the age of the infected host [START_REF] Tucker | Viral determinants of age-dependent virulence of Sindbis virus for mice[END_REF]. Moreover, it was shown that the NSV strain induces high mortality rates in both weanling and adult mice,

showing how different strains can result in very different infectivity and lethality. This is also well exemplified by the comparison of our two SINV strains (SINV-1GFP and SINV-2mCherry).

Importantly, even though it was reported that SINV persists in a non-replicative form in the CNS of mice that recovered from acute encephalitis [START_REF] Levine | Persistence of viral RNA in mouse brains after recovery from acute viral encephalitis[END_REF], it was also shown that SINV can induce apoptosis, and this correlates with its neurovirulence (Lewis et al., 1996). This phenomenon was also observed in zebrafish larvae, in particular those displaying an extensive infection in the CNS. However, we will need to quantify precisely the level of apoptosis in correlation with the level of CNS infection, also with the help of our newly established zebrafish neuronal cell cultures. SINV-induced death of neurons has been shown to be limited by autophagy in a cell-autonomous manner, in elegant in vivo experiments in mice [START_REF] Orvedahl | Autophagy protects against Sindbis virus infection of the central nervous system[END_REF]. This is something that we could also tackle in zebrafish, in collaboration with Emma Colucci-Guyon who developed tools to manipulate autophagy in the context of bacterial infections (Mostowy et al., 2013 and unpublished results).

Although in the majority of studies conducted in mice SINV is inoculated ic, Cook and Griffin have shown how the use of a luciferase-expressing SINV can be used to monitor infection after subcutaneous injection (Cook and Griffin, 2003),

and have suggested entry in the CNS via retrograde axonal transport. Importantly, they have also shown how the variability of the infection between different mice is not exclusively dependent on the mouse strain. Different mice of the same strain developed in fact infection in different areas (nose, spinal cord, brain) and at different times post-inoculation. In our study, we have also observed a high variability in terms of number of larvae with CNS infection and patterns of infection in the brain. Therefore, these differences can be attributed to intrinsic characteristics of the virus rather than to the genetic heterogeneity of the zebrafish.

General Perspective: Pride and Prejudice of In Vivo Models

"Remember that all models are wrong; the practical question is how wrong do they have to be to not be useful."

Box, G.E.P., Draper, N.R., (1987). Empirical Model Building and Response Surfaces. John Wiley and Sons, New York, NY., p. 74.

According to the British mathematician George E.P. Box (1919Box ( -2013)), all models are intrinsically wrong, being a simplified representation of an infinitely complex truth. Although perhaps discouraging at first, this concept should instead be the driving force leading researchers into finding the most appropriate model to answer their scientific question, thereby turning something "wrong" into something useful.

Often no non-animal alternative is feasible (e.g. in vitro cell cultures, in silico modeling); therefore, we must carefully choose the appropriate animal model for the validity of our research. According to Davidson et al., the selection of animal models should be based on several considerations [START_REF] Davidson | Requirements and selection of an animal model[END_REF]):

1. Appropriateness as an analog 2. Genetic uniformity of organisms, where applicable 3. Transferability of information, in particular in the case of disease modeling 4. Generalizability of the results, in particular for pharmacological and toxicological modeling 5. Background knowledge of biological properties 6. Ethical implications, by justifying the use of animal models 7. Ease of and adaptability to experimental manipulation 8. Costs and availability

Ecological consequences

With all these criteria in mind, it seems natural that only a limited number of animal models is widely accepted and largely supported by funding agencies. The mouse (Mus musculus), the nematode worm (Caenorhabditis elegans), the fruitfly (Drosophila melanogaster) and the thale cress (Arabidopsis thaliana) represent for many experimental biologists the only acceptable models. Often, submitting a grant proposal using a standard organism does not necessitate explaining the chosen model. In contrast, choosing a less common one requires detailed justifications to convince sceptical and more conservative colleagues.

However, a usually widely accepted organism can turn out to be the wrong one, because of wrong initial assumptions. In the case of drug discovery, we find an increasing number of examples where potential drugs fail to pass Phase II clinical trials due to lack of efficacy or toxicity, despite being proven safe and effective in preclinical models. Inbred mice administered with an experimental treatment for multiple sclerosis, for example, were shown to improve from the induced disease while the same treatment in human patients gave unpredicted and/or adverse responses during Phase II trials [START_REF] Kappos | Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial. The altered peptide ligand in relapsing MS study group[END_REF]. It is clear that for certain immunological studies, inbred mice fail at mimicking the appropriate level of complexity and diversity of human genetics.

In other cases, we fail at choosing the appropriate number of animals to detect a significant effect, either because of the costs, of the limited availability of the animals, of errors in experimental setup, or for the pressure of reducing the number of animals used. Although, one of the tasks of the Ethical Committees is to make sure that an appropriate number of animals is used (not too high, but not too low either as to become useless for statistical analyses).

After these considerations, where does the zebrafish fit in the mayhem of accepted and acceptable model organisms? Its optical clarity, ease of genetic manipulations, and small size at the larval stage have always been regarded as major advantages over other vertebrate models. However, when used for drug screenings, we must keep in mind the differences in body size, stage of development and, potentially, in metabolic patterns between fish and humans to find the correct dose response curve.

With these premises in mind, the high number of eggs per clutch, the ease of screening for specific phenotypes, and the small amount of compound needed, make the zebrafish a valuable model to test activity and toxicity of a large number of potential therapeutics, in a short time frame and with reduced costs. Our experiments to validate the antiviral effects of DFMO in zebrafish larvae are a clear example of a fast and relatively cheap in vivo drug test that was much more difficult and limited in mouse due to the elevated costs. Moreover, behavioral studies in the context of compound toxicity investigations are also becoming common practice in zebrafish and could be included in disease modeling studies, to obtain more exhaustive data with respect to the observed clinical signs. Another major advantage consists in the possibility to use algorithms that extract meaningful data from images, which greatly help reducing the time required to screen the efficacy of a compound in hundreds of fish. All these points make the zebrafish a good model for translational research.

In this work, we have shown how the combined use of fluorescent recombinant viral strains and zebrafish transgenic lines allow for the investigation of a virus infection course from early to late times post-infection. At the same time, we have been able to follow the elicited immune response and to correlate it with the levels of infection observed in single larvae. We have shown that the zebrafish and the mouse model can provide comparable information, suggesting we should apply inter-species approaches to validate our respective models of infection. We have shown how zebrafish larvae can be employed in drug screening tests, providing important results at reduced costs and with less time required for the analysis. I hope that with this and future works, we will continue to show how the zebrafish can represent a useful model to understand virus-induced pathogenesis and to find safe and valuable therapeutics.

MATERIALS AND METHODS

MATERIALS AND METHODS

This section provides a detailed description of the protocol used to produce SINV pTR339-mCherry/2A and SINV pTR339-GFP/2A, as well as the protocol set up to obtain zebrafish neuronal cells in culture. All other methods have been previously described or included in the "Materials and Methods" section of the submitted article, and therefore will not be described further.

Sindbis Virus Production and Titration on BHK cells

The following protocol was kindly provided by Benjamin Obadia and Carla Saleh and has been adapted from Hardwick and Levine (2000). It consists in the linearization, in vitro transcription and transfection of SINV plasmids, in order to produce new concentrated virus stocks. The volume is dependent on the Ibidi dish being used (1 mL/dish for the Ibidi µ-Dish 35 mm, high and the Ibidi Hi-Q 4 , 150 µL/well for the Ibidi µ-Slide 8 well )

3. Remove PDL and wash 3x with PBS 4. Remove PBS and store the dishes at room temperature On the day of the dissociation:

The following passages must be performed under a laminar flow hood, to avoid contaminations of the cell culture Head Dissociation 1. Place ∼100-150 72 hpf zebrafish embryos, still in their chorions, in EtOH 70%

(5-10 sec)

2. Quickly remove the embryos, using a sterile pipette and wash them 2x with sterile water with methylene blue

BIBLIOGRAPHY Introduction

Teleosts, the largest and best-known clade of ray-finned fish, constitute a highly successful and diverse group, including half of vertebrate species. Their line and ours diverged about 450 million years ago. Several species within this group, both commercial species and model organisms, have been studied to some depth by immunologists, and many details of their antiviral defenses are now known. Although fish genomes have a complex history of whole genome duplications (WGDs) and contractions, the remarkable conservation of the interferon (IFN) system underlines the critical importance of innate antiviral immunity in vertebrates. Extensive studies performed in mammals in various contexts of viral infection demonstrated the importance of IFNs in antiviral responses. The name of this group of cytokines originates in their ability to "interfere" with the viral progression, as first described in 1957 by Isaacs and Lindenmann [START_REF] Isaacs | Virus interference. I. The interferon[END_REF]. IFNs belong to class II helical cytokine family and, in mammals, can be divided into three different groups based on biological and structural features as well as receptor usage [START_REF] Pestka | Interferons, interferon-like cytokines, and their receptors[END_REF]: mammalian IFNs have been classified as type I (α, β, ω, ε, and κ), type II (γ), and type III (λ) IFNs. Actually, only type I and type III IFNs (often grouped under the label "virus-induced IFNs") are truly specialized as innate antiviral cytokines; IFNγ is rather a regulatory cytokine of innate and adaptive immunity, mostly active against intracellular bacteria.

IFN-like antiviral activity has been reported in fish 40 years ago [START_REF] De Kinkelin | Interferon production in rainbow trout (Salmo gairdneri) experimentally infected with Egtved virus[END_REF][START_REF] Oie | Reovirus type 2: induction of viral resistance and interferon production in fathead minnow cells[END_REF]. However, teleost IFN genes could not be identified before the development of fish genomics [START_REF] Robertsen | Atlantic salmon interferon genes: cloning, sequence analysis, expression, and biological activity[END_REF][START_REF] Altmann | Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio[END_REF][START_REF] Lutfalla | Comparative genomic analysis reveals independent expansion of a lineage-specific gene family in vertebrates: the class II cytokine receptors and their ligands in mammals and fish[END_REF][START_REF] Zou | Teleost fish interferons and their role in immunity[END_REF]. These virus-induced fish IFNs were clearly responsible for a strong inducible activity against a range of viruses [START_REF] Robertsen | Atlantic salmon interferon genes: cloning, sequence analysis, expression, and biological activity[END_REF][START_REF] Altmann | Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio[END_REF][START_REF] Lutfalla | Comparative genomic analysis reveals independent expansion of a lineage-specific gene family in vertebrates: the class II cytokine receptors and their ligands in mammals and fish[END_REF]. Although some fish species (e.g., fugu or medaka) appear to possess one single virus-induced IFN gene, the number of identified genes grew rapidly in other fish species. There are four virus-induced IFN genes in zebrafish (aka IFNφ) [START_REF] Zou | Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates[END_REF][START_REF] Aggad | The two groups of zebrafish virusinduced interferons signal via distinct receptors with specific and shared chains[END_REF], a number unlikely to change much considering the quality reached by the zebrafish genome assembly. Salmonids, however, have many more IFN genes; the current record is 11 genes in Atlantic salmon [START_REF] Sun | Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties[END_REF]. Two main subsets could be distinguished among fish virus-induced IFNs, corresponding to the number of cysteine (C) residues predicted to be engaged in disulfide bridges: two for IFNs of group I and four for IFNs of group II [START_REF] Zou | Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates[END_REF][START_REF] Sun | Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties[END_REF], as was later confirmed by three-dimensional crystallography [START_REF] Hamming | Crystal structure of Zebrafish interferons I and II reveals conservation of type I interferon structure in vertebrates[END_REF]. The 4C configuration is found in all tetrapod type I IFNs, with the exception of mammalian IFNβ, which has only one disulfide bridge. However, the cysteine pair of IFNβ is different from the one of fish group I IFNs, and one should emphasize that the two groups of fish IFNs do not correspond to the alpha/beta subdivision of mammalian type I IFNs, which occurred after the divergence of avian and mammalian lineages.

Two different isoforms of some fish IFN transcripts, resulting from the usage of alternative promoters, show different levels of induction: upon viral infection, a short transcript encoding a protein with a signal peptide is induced in addition to a constitutively expressed isoform, which lacks signal peptide [START_REF] Levraud | Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system[END_REF]. This particularity has been observed in a number of fish species, but not for all their IFN genes [START_REF] Bergan | Promoters of type I interferon genes from Atlantic salmon contain two main regulatory regions[END_REF][START_REF] Long | Identification and expression analysis of cDNAs encoding channel catfish type I interferons[END_REF][START_REF] Purcell | Infectious haematopoietic necrosis virus genogroup-specific virulence mechanisms in sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake, Idaho[END_REF]. No function of the presumably non-secreted IFN isoform, unique to teleosts as far as we know, has been reported.

Importantly, the two groups of IFNs were found to signal via two different receptors in zebrafish (Fig. 1) [START_REF] Aggad | The two groups of zebrafish virusinduced interferons signal via distinct receptors with specific and shared chains[END_REF]. IFNφs of the first group (IFNφ1 and φ4) bind to the cytokine receptor family B (CRFB)1-CRFB5 complex while the CRFB5 chain is associated to CRFB2 to form the receptor for group II (IFNφ2 and φ3) [START_REF] Levraud | Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system[END_REF][START_REF] Aggad | The two groups of zebrafish virusinduced interferons signal via distinct receptors with specific and shared chains[END_REF]. Interestingly, both zebrafish IFNφ4 and salmon IFNd-which are possible orthologuesseem to have lost antiviral activity and might be on their way becoming pseudogenes. Alternatively, they may even play a decoy role for other IFNs.

Do the two groups of fish virus-induced fish IFNs play distinct or redundant roles? By injecting recombinant IFNs in adult zebrafish and challenging them with different pathogens, Lopez-Munoz et al. found that both types would protect against a virus, but only the group I IFN would also protect against a bacteria [START_REF] Lopez-Munoz | New insights into the evolution of IFNs: activities genes and display powerful antiviral transient expression of IFN-dependent zebrafish group II IFNs induce a rapid and display powerful antiviral activities[END_REF]; they also observed an induction of distinct gene subsets. However, it is difficult to reach a firm conclusion from this study, because untitered culture supernatants were used as sources of recombinant IFNs, and because the slow kinetics of induction of most downstream genes (including the IFN themselves) suggests indirect effects. Most other studies found quantitative but not clearly qualitative differences between the responses induced by the different IFNs (e.g., Ref. [START_REF] Svingerud | Atlantic salmon type I IFN subtypes show differences in antiviral activity and cell-dependent expression: evidence for high IFNb/IFNc-producing cells in fish lymphoid tissues[END_REF]), although this remains to be analyzed in depth. Nevertheless, the distinct receptors for the two IFN groups raise the possibility of different target tissues; in addition, important differences in expression patterns of the different fish IFNs have been demonstrated. The spatial differences of IFN and interferon-stimulated gene (ISG) expression will be reviewed in later sections.

Classification of virus-induced fish IFN genes, relative to mammalian IFNs, has been controversial for some time. Molecular phylogenies were uncertain because the low overall similarity (b 25%) between mammalian and fish proteins resulted in uncertain software-generated alignments. It was thus not possible to claim with certainty that fish virus-induced IFNs were closer to mammalian type I or type III IFNs (or co-orthologous to both groups as a set of paralogues), although some sequence features, such as the CAWE sequence at the beginning of the C-terminal helix, were noted by some as characteristic of type I IFNs [START_REF] Zou | Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates[END_REF][START_REF] Sun | Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties[END_REF][START_REF] Robertsen | The interferon system of teleost fish[END_REF]. By contrast, fish IFN genes are composed of five exons and four introns [START_REF] Sun | Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties[END_REF][START_REF] Robertsen | The interferon system of teleost fish[END_REF], as are mammalian type III IFN genes, while mammalian type I IFN genes contain a single exon; additionally, when receptors for IFNs were identified in zebrafish, their domain organization had features of the receptor of human IFN λ rather than type I IFN receptor, which has a uniquely large extracellular region in one chain (Fig. 1) [START_REF] Levraud | Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system[END_REF]. However, the first argument was soon dismissed when frogs were found to have both type I and type III IFNs, all with five-exon structures, indicating that single-exon type I IFN genes were the result of a retrotransposition event in the amniote lineage, not an ancestral feature [START_REF] Qi | Intron-containing type I and type III IFN coexist in amphibians: refuting the concept that a retroposition event gave rise to type I IFNs[END_REF]. Finally, crystal structures revealed a characteristic type I IFN architecture for both groups of IFNφs with a straight F helix, as opposed to the remaining class II cytokines, including IFN-λ, where helix F is bent [START_REF] Hamming | Crystal structure of Zebrafish interferons I and II reveals conservation of type I interferon structure in vertebrates[END_REF].

Based on these considerations, different names have been proposed for fish IFNs: type I IFNs, virus-induced IFNs, IFNλ, or even simply IFNs. Following Stein et al. [21], zebrafish IFNs are now called IFNφ (φ for fish). While it is now demonstrated that fish virus-induced IFNs are structurally type I IFNs, a consensus about a consistent nomenclature for these cytokines has still to be reached. The current zebrafish nomenclature avoids orthology assumptions but does not clearly distinguishes group I and group II IFNs. The current nomenclature for salmonid IFNs, which groups the genes into four subgroups, IFNa, IFNb, IFNc, and IFNd [START_REF] Sun | Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties[END_REF][START_REF] Zou | Origin and evolution of the RIG-I like RNA helicase gene family[END_REF], has the same issue (group 1 includes IFNas and IFNds; group 2 includes IFNbs and IFNcs) with the caveat that unaware readers could wrongly assume that IFNas are orthologous to mammalian IFNαs, and IFNbs to IFNβ. A self-explanatory nomenclature reflecting the phylogenetic relationships between IFN genes remains to be established.

Fish also possess clear orthologues of mammalian type II IFNs (γ), with many fish species having two type II ifn genes (ifnγ1 and ifnγ2) [START_REF] Long | Identification and expression analysis of cDNAs encoding channel catfish type I interferons[END_REF][START_REF] Zou | Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish[END_REF][START_REF] Sieger | The role of gamma interferon in innate immunity in the zebrafish embryo[END_REF][START_REF] Stolte | Differential expression of two interferon-gamma genes in common carp (Cyprinus carpio L.)[END_REF]. In zebrafish, IFNγ1 and IFNγ2 bind to distinct receptors: the IFNγ2 receptor includes Crfb6 together with CRFB13 and CRFB17, while the IFNγ1 receptor does not comprise CRFB6 or CRFB13 but includes CRFB17 (Fig. 1) [START_REF] Aggad | In vivo analysis of Ifn-γ1 and Ifn-γ2 signaling in zebrafish[END_REF]. Genes encoding a trout receptor of IFNγ have also been identified [START_REF] Gao | The search for the IFN-g receptor in fish: functional and expression analysis of putative binding and signalling chains in rainbow trout Oncorhynchus mykiss[END_REF]. Infection studies show that IFNγ signaling is involved in resistance against bacterial infections in the zebrafish embryo, with a proper level required for the fish to clear high doses of Escherichia coli or low doses of the fish pathogen Yersinia ruckeri [START_REF] Sieger | The role of gamma interferon in innate immunity in the zebrafish embryo[END_REF]. However, a potent antiviral activity of IFNγ was also demonstrated in Atlantic salmon against infectious pancreatic necrosis virus (IPNV) and infectious salmon anemia virus (ISAV), which may partly depend on the coexpression of type I IFN [START_REF] Sun | Antiviral activity of salmonid gamma interferon against infectious pancreatic necrosis virus and salmonid alphavirus and its dependency on type I interferon[END_REF]. However, fish IFNγ are not always induced by viral infections under conditions where type I IFNs are [START_REF] Aggad | In vivo analysis of Ifn-γ1 and Ifn-γ2 signaling in zebrafish[END_REF], indicating that in fish as well as in mammals, IFNγ are probably not specialized antiviral cytokines; they will therefore not be discussed further.

Virus sensors in fish and their signaling pathways

In mammals, viral infection is rapidly detected by specialized PRRs (pattern recognition receptors) such as RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs). These cellular sensors of invading pathogens are directly involved in the activation of the IFN system.

Three RLRs, that is, RNA helicases containing canonical DExD/H motifs, have been identified to date in humans: retinoic acid-inducible gene I (RIG-I, also known as DDX58), melanoma differentiation-associated gene 5 (MDA5, or IFIH1), and laboratory of genetics and physiology 2 (LGP2, or DHX58). In silico analyses led to the identification of RLRs described in many teleost fish including zebrafish, Atlantic salmon, grass carp, Japanese flounder, rainbow trout, and fathead minnow [START_REF] Zou | Origin and evolution of the RIG-I like RNA helicase gene family[END_REF][START_REF] Biacchesi | Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses[END_REF][START_REF] Lauksund | Atlantic salmon IPS-1 mediates induction of IFNa1 and activation of NF-k B and localizes to mitochondria[END_REF][START_REF] Huang | Identification and expression profiling analysis of grass carp Ctenopharyngodon idella LGP2 cDNA[END_REF][START_REF] Ohtani | Evolutional conservation of molecular structure and antiviral function of a viral RNA receptor, LGP2, in Japanese flounder, Paralichthys olivaceus[END_REF][START_REF] Ohtani | Characterization and antiviral function of a cytosolic sensor gene, MDA5, in Japanese flounder, Paralichthys olivaceus[END_REF][START_REF] Simora | Molecular cloning and antiviral activity of IFN-β promoter stimulator-1 (IPS-1) gene in Japanese flounder, Paralichthys olivaceus[END_REF][START_REF] Chang | Expression and functional characterization of the RIG-I-like receptors MDA5 and LGP2 in Rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Yang | Identification of a retinoic acid-inducible gene I from grass carp (Ctenopharyngodon idella) and expression analysis in vivo and in vitro[END_REF]. These sequences are highly conserved between mammalian and fish orthologues [START_REF] Rajendran | Pathogen recognition receptors in channel catfish: II. Identification, phylogeny and expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)[END_REF]. LGP2 and MDA5 seem to be conserved in all fish species, while RIG-I has been retrieved only in some groups including salmonids and cyprinids [START_REF] Hansen | Sensing disease and danger: a survey of vertebrate PRRs and their origins[END_REF]. Like their mammalian counterparts, expression of RLRs is modulated upon viral infection [START_REF] Biacchesi | Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses[END_REF][START_REF] Huang | Identification and expression profiling analysis of grass carp Ctenopharyngodon idella LGP2 cDNA[END_REF][START_REF] Ohtani | Evolutional conservation of molecular structure and antiviral function of a viral RNA receptor, LGP2, in Japanese flounder, Paralichthys olivaceus[END_REF][START_REF] Yang | Identification of a retinoic acid-inducible gene I from grass carp (Ctenopharyngodon idella) and expression analysis in vivo and in vitro[END_REF][START_REF] Biacchesi | Both STING and MAVS fish orthologs contribute to the induction of interferon mediated by RIG-I[END_REF][START_REF] Su | Molecular cloning and immune responsive expression of MDA5 gene, a pivotal member of the RLR gene family from grass carp Ctenopharyngodon idella[END_REF] and IFN stimulation through polyI: C treatment [START_REF] Ohtani | Characterization and antiviral function of a cytosolic sensor gene, MDA5, in Japanese flounder, Paralichthys olivaceus[END_REF] or by ubiquitin-like ISG15 [START_REF] Langevin | Zebrafish ISG15 exerts a strong anti-viral activity against RNA and DNA viruses and regulates the interferon response[END_REF], which also modulates RIG-I activity [START_REF] Kim | Negative feedback regulation of RIG-I-mediated antiviral signaling by interferon-induced ISG15 conjugation[END_REF]. Interestingly, LGP2 appears to be a positive activator of the IFN pathway in fish. Sequence analysis suggests a fair conservation of signaling pathways downstream of RLR (Fig. 2), with a critical role of for the mitochondrial antiviral signaling protein (MAVS, also known as CARDIF, VISA, or IPS-1) [START_REF] Zou | Origin and evolution of the RIG-I like RNA helicase gene family[END_REF][START_REF] Biacchesi | Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses[END_REF][START_REF] Simora | Molecular cloning and antiviral activity of IFN-β promoter stimulator-1 (IPS-1) gene in Japanese flounder, Paralichthys olivaceus[END_REF][START_REF] Su | Molecular cloning, characterization and expression analysis of interferon-β promoter stimulator 1 (IPS-1) gene from grass carp Ctenopharyngodon idella[END_REF][START_REF] Xiang | Characterization of a TnMAVS protein from Tetraodon nigroviridis[END_REF]. Association of MAVS with TRAF [tumor necrosis factor (TNF) receptor-associated factor] 3 and activation of the pathway by TBK1 (TANK binding kinase 1) via phosphorylation of IFN regulatory factor (IRF)3/7 transcriptional factors have also been shown in fish [START_REF] Xiang | Characterization of a TnMAVS protein from Tetraodon nigroviridis[END_REF][START_REF] Sun | Characterization of fish IRF3 as an IFN-inducible protein reveals evolving regulation of IFN response in vertebrates[END_REF]. Nuclear translocation of these factors induced the transcription of different cytokines including IFN genes. The adaptor STING (aka "mediator of IRF3 activation" or MITA, ERIS, and MYPS), a transmembrane protein located in the endoplasmic reticulum, links signaling between MAVS and downstream cytosolic kinase TBK1 [START_REF] Zhong | The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation[END_REF][START_REF] Ishikawa | STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling[END_REF]. In mammals, STING is also involved in the induction of IFNβ by DNA viruses, connecting cytosolic DNA sensing to TBK1 and IRF3 activation [START_REF] Ishikawa | STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity[END_REF]. STING has been identified in fish and plays an important role in the RLR/ IRF3-dependent signaling [START_REF] Biacchesi | Both STING and MAVS fish orthologs contribute to the induction of interferon mediated by RIG-I[END_REF][START_REF] Sun | Fish MITA serves as a mediator for distinct fish IFN gene activation dependent on IRF3 or IRF7[END_REF]. The pathways induced by DNA viruses are still poorly known in fish, and the importance of STING in this signaling remains to be established. Interestingly, the DNA sensors AIM2 and IFI6-16 seem to be missing in fish.

A diverse TLR repertoire has been found in fish [START_REF] Roach | The evolution of vertebrate Toll-like receptors[END_REF][START_REF] Palti | Toll-like receptors in bony fish: from genomics to function[END_REF]. Some TLRs have been described only in lower vertebrates including TLR14 and TLR23 [START_REF] Roach | The evolution of vertebrate Toll-like receptors[END_REF]; TLR18, TLR19, and TLR20 [START_REF] Meijer | Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish[END_REF]; TLR21 and TLR22 [START_REF] Oshiumi | Prediction of the prototype of the human Toll-like receptor gene family from the pufferfish, Fugu rubripes, genome[END_REF]; TLR24 [START_REF] Kasamatsu | Phylogenetic and expression analysis of lamprey toll-like receptors[END_REF]; and TLR25 and TLR26 [START_REF] Quiniou | Comprehensive survey and genomic characterization of Toll-like receptors (TLRs) in channel catfish, Ictalurus punctatus: identification of novel fish TLRs[END_REF]. TLRs, which are involved in the recognition of doublestranded RNA (dsRNA) (TLR3) or single-stranded RNA (TLR7 and 8) in mammals, have good orthologues in fish [START_REF] Meijer | Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish[END_REF][START_REF] Oshiumi | Prediction of the prototype of the human Toll-like receptor gene family from the pufferfish, Fugu rubripes, genome[END_REF][START_REF] Jault | Toll-like receptor gene family and TIR-domain adapters in Danio rerio[END_REF]. Both structural and functional evidence indicate that these TLR are also involved in virus sensing in fish: all critical residues for binding to dsRNA are conserved in fish TLR3 [START_REF] Quiniou | Comprehensive survey and genomic characterization of Toll-like receptors (TLRs) in channel catfish, Ictalurus punctatus: identification of novel fish TLRs[END_REF], and RTG-2 rainbow trout cells transfected with TLR3 showed increased IFN response after poly(I: C) stimulation [START_REF] Matsuo | Teleost TLR22 recognizes RNA duplex to induce IFN and protect cells from birnaviruses 1[END_REF]. Similarly, the leucin-rich repeats of TLR7 are remarkably conserved between mammals and fish [START_REF] Quiniou | Comprehensive survey and genomic characterization of Toll-like receptors (TLRs) in channel catfish, Ictalurus punctatus: identification of novel fish TLRs[END_REF][START_REF] Mikami | Molecular evolution of vertebrate Toll-like receptors: evolutionary rate difference between their leucine-rich repeats and their TIR domains[END_REF], and a known ligand of TLR7 and TLR8 (R-848) induces a typical IFN response in salmonid leukocytes [START_REF] Svingerud | Atlantic salmon type I IFN subtypes show differences in antiviral activity and cell-dependent expression: evidence for high IFNb/IFNc-producing cells in fish lymphoid tissues[END_REF][START_REF] Palti | Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss)[END_REF]. Additionally, among fish-specific TLRs, TLR22 is responsive to virus infections, poly(I:C), and dsRNA [START_REF] Matsuo | Teleost TLR22 recognizes RNA duplex to induce IFN and protect cells from birnaviruses 1[END_REF][START_REF] Hirono | Characterization of gene structure and expression of two toll-like receptors from Japanese flounder, Paralichthys olivaceus[END_REF]. Fugu TLR22 recognizes long-sized dsRNA on the cell surface, while TLR3 binds short-sized dsRNA in the endoplasmic reticulum [START_REF] Matsuo | Teleost TLR22 recognizes RNA duplex to induce IFN and protect cells from birnaviruses 1[END_REF], which may represent a dual pathway for RNA virus sensing in fish.

Upon ligand binding, TLRs dimerize and their intracytoplasmic TIR (Toll-interleukin 1 receptor) domains recruit adaptor molecules through homotypic TIR/TIR interactions. In mammals, most TLRs signal through the Myd88 adaptor, which recruits interleukin-1R-associated kinase (IRAK) (Fig. 2). This protein then associates with TRAF6, subsequently involving TANK (TRAF family memberassociated NfkB activator kinase) and IKKi (inhibitor of NFkB kinase) inducing NFκB nuclear translocation and type I IFN gene transcription. In contrast, TLR3 (specific for dsRNA) signaling occurs independently of Myd88 through the recruitment of TRIF (TIR domain containing adaptor inducing IFNβ, also known as TICAM-1 or Myd88-3), leading to TRAF3 signaling cascade, IRF3 phosphorylation preceding nuclear translocation, and recognition of IFNstimulated responses elements on type I IFN promoters. Viral infection alternatively activates IRF7 via TLR7-9 in a TRAF6-dependent manner [START_REF] Kawai | The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors[END_REF]. Although TLR families show distinct features among vertebrates, the components of signaling pathways are well conserved as suggested by the presence of kinase and adaptor molecule orthologues in zebrafish and pufferfish [START_REF] Stein | Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish[END_REF]. Myd88 and other TIR adaptors were identified in zebrafish [START_REF] Jault | Toll-like receptor gene family and TIR-domain adapters in Danio rerio[END_REF], and morpholino approaches as well as infectious models demonstrated the functionality of Myd88 in the establishment of TLR-mediated immune response [START_REF] Van Der Sar | MyD88 innate immune function in a zebrafish embryo infection model[END_REF]. Further studies confirmed these observations using different stimulations [poly(I:C), flagelin, or chemical treatments] [START_REF] Takano | Molecular cloning and characterization of Toll-like receptor 9 in Japanese flounder, Paralichthys olivaceus[END_REF][START_REF] Purcell | Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus[END_REF]. Since then, myd88 has been identified in many fish species [START_REF] Purcell | Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus[END_REF][START_REF] Yao | Molecular cloning and expression of MyD88 in large yellow croaker, Pseudosciaena crocea[END_REF][START_REF] Rebl | Salmonid Tollip and MyD88 factors can functionally replace their mammalian orthologues in TLR-mediated trout SAA promoter activation[END_REF][START_REF] Yu | Full-length sequence and expression analysis of a myeloid differentiation factor 88 (MyD88) in half-smooth tongue sole Cynoglossus semilaevis[END_REF][START_REF] Skjaeveland | Identification and characterization of TLR8 and MyD88 homologs in Atlantic salmon (Salmo salar)[END_REF]. Zebrafish TRIF similarly triggered activation of type I IFN. The TRIF-dependent TLR pathway converges with the RLR pathway by activating the TBK1 kinase, which is conserved in fish as mentioned above. However, the TICAM1 signaling pathway observed in zebrafish is apparently independent of IRF3 and IRF7 and does not require interaction with TRAF6 [START_REF] Sullivan | Evidence for evolving Toll-IL-1 receptor-containing adaptor molecule function in vertebrates[END_REF]. Also, a gene coding for the IRAK2 kinase is missing from the genome of pufferfish, zebrafish, medaka, and stickleback [START_REF] Stein | Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish[END_REF], while an IRAK1 orthologue is present and can trigger innate immune response [START_REF] Rebl | Toll-like receptor signaling in bony fish[END_REF].

Thus, IFN-inducing signaling pathways are overall fairly well conserved between fish and mammals. Regarding the sensors, RLRs are also remarkably well conserved, while the fish TLR repertoire include a variety of receptors absent in mammals-some of which, at least, contribute to viral detection-in addition to well-conserved ones such as TLR3 and TLR7.

Conserved signaling pathways downstream of IFN receptors

In mammals, IFN binding to their membrane receptors leads to the activation of the JAK-STAT signaling pathway (Fig. 1). Type I IFN association to its receptor triggers recruitment and binding of the kinases TYK2 and JAK1 to IFNAR1 and IFNAR2, respectively. Subsequently, these kinases promote the phosphorylation of STAT1 and STAT2 proteins preceding their oligomerization. Conjugation of cytoplasmic IRF9 to the STAT1/2 oligomers generates the complex ISGF3 (IFN-stimulated gene factor), which induces the transcription of ISGs after binding nuclear IFN-stimulated responses elements on their promoter. In fish, the stat1 gene has been described in many species [START_REF] Yu | Full-length sequence and expression analysis of a myeloid differentiation factor 88 (MyD88) in half-smooth tongue sole Cynoglossus semilaevis[END_REF][START_REF] Collet | Infectious pancreatic necrosis virus suppresses type I interferon signalling in rainbow trout gonad cell line but not in Atlantic salmon macrophages[END_REF][START_REF] Park | Molecular cloning and expression analysis of the STAT1 gene from olive flounder, Paralichthys olivaceus[END_REF][START_REF] Skjesol | Structural and functional studies of STAT1 from Atlantic salmon (Salmo salar)[END_REF]; the zebrafish genome encodes two different paralogues, stat1a and stat1b [START_REF] Stein | Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish[END_REF]. Functional studies highlighted their role in the regulation of the type I IFN pathway in different species [START_REF] Yu | Full-length sequence and expression analysis of a myeloid differentiation factor 88 (MyD88) in half-smooth tongue sole Cynoglossus semilaevis[END_REF][START_REF] Collet | Infectious pancreatic necrosis virus suppresses type I interferon signalling in rainbow trout gonad cell line but not in Atlantic salmon macrophages[END_REF][START_REF] Skjesol | Structural and functional studies of STAT1 from Atlantic salmon (Salmo salar)[END_REF]. However, the respective roles of the different STAT1s in IFN pathway regulation remain unclear in zebrafish. Kinases JAK1 and TyK2 as well as STAT2 and IRF9 are also present in fish genomes [START_REF] Stein | Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish[END_REF]. Aggad et al. proposed that TYK2 would be associated to CRFB5, while JAK1 would be associated to CRFB1 and 2, thus leading to the activation of the IFN signaling pathway and to viperin transcription (Fig. 2) [START_REF] Aggad | The two groups of zebrafish virusinduced interferons signal via distinct receptors with specific and shared chains[END_REF].

In contrast, type II IFNs signal after binding to IFNGR1-2 by recruiting JAK1 and JAK2; these kinases promote phosphorylation of STAT1 homodimer, which directly translocates to the nucleus and bind a GAS element (IFN gamma-activated site), thus mediating up-regulation of a broad repertoire of genes, partly overlapping with the type I IFN-mediated response. In zebrafish, IFN-γ1 and IFN-γ2 bind distinct receptors (CRFB6-CRFB13 and CRFB17 for IFN-γ2 and CRFB17, plus unidentified chains, for IFN-γ1) with conserved binding regions of JAK1 and 2 kinases [START_REF] Aggad | In vivo analysis of Ifn-γ1 and Ifn-γ2 signaling in zebrafish[END_REF]. Two JAK2 kinases are expressed in this species (JAK2a and b), and only JAK2a has been involved in IFNγ signaling using constitutively active mutants (Figs. 1 and2) [START_REF] Aggad | In vivo analysis of Ifn-γ1 and Ifn-γ2 signaling in zebrafish[END_REF]. Future studies will be required to determine which of the two STAT1 paralogues constitutes the active protein involved in the signaling pathway of type I and type II IFNs.

Part 2. ISGs and Their Diverse Evolutionary Patterns

Type I IFNs do not possess antiviral activity per se but interfere with viral infection through induction of a vast repertoire of ISGs via the JAK/STAT pathway. A few hundred ISGs have been identified in human [START_REF] Sadler | Interferon-inducible antiviral effectors[END_REF][START_REF] Schoggins | A diverse range of gene products are effectors of the type I interferon antiviral response[END_REF], with a rich diversity of molecular functions. Some ISGs exert a direct antiviral activity such as MX, VIPERIN/ VIG1, ISG15, PKR, and TRIM5. However, the connection of most ISGs to antiviral mechanisms, and even their role in the biology of the cell, remain unknown.

While ISGs are intrinsically located downstream of IFN in the antiviral pathways induced by viral infections, a number of them are able to up-regulate type I IFNs and are therefore involved in positive feedback regulatory loops (e.g., trim25, rigI, stat1, irf7, and viperin/vig1 [START_REF] Gack | TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity[END_REF][START_REF] Yoneyama | The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses[END_REF][START_REF] Honda | IRF-7 is the master regulator of type-I interferondependent immune responses[END_REF][START_REF] Saitoh | Antiviral protein Viperin promotes Toll-like receptor 7-and Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells[END_REF], while some also feedback negatively on IFN signaling (e.g., socs1 and 2). Furthermore, the recognition of viral compounds by cellular sensors can up-regulate some ISGs directly, that is, independently of IFN induction; such bypass has been shown for example for Mx [START_REF] Goetschy | Regulation of the interferon-inducible IFI-78K gene, the human equivalent of the murine Mx gene, by interferons, double-stranded RNA, certain cytokines, and viruses[END_REF][START_REF] Dewitte-Orr | Induction of antiviral genes, Mx and vig-1, by dsRNA and Chum salmon reovirus in rainbow trout monocyte/macrophage and fibroblast cell lines[END_REF] and for viperin in human and fish [START_REF] Zhu | Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: induction of interferon-responsive RNAs[END_REF][START_REF] Boudinot | vig-1, a new fish gene induced by the rhabdovirus glycoprotein, has a virus-induced homologue in humans and shares conserved motifs with the MoaA family[END_REF]. Hence, while IFN definitely plays a central role in the innate antiviral response, a complex and redundant network of regulatory loops and bypass mechanisms is also involved, which makes the whole system more resistant to subversion by viruses.

Orthologues of human ISGs involved in IFN amplification have often been retrieved as ISGs in fish, which may indicate that they belong to the primordial IFN pathway: for example, trim25, rigI, stat1, irf7, and viperin/vig1 are conserved in teleost fish and are induced by type I IFN in these organisms [START_REF] O'farrell | Survey of transcript expression in rainbow trout leukocytes reveals a major contribution of interferon-responsive genes in the early response to a rhabdovirus infection[END_REF]. In fish, this list includes also irf3 [START_REF] Sun | Characterization of fish IRF3 as an IFN-inducible protein reveals evolving regulation of IFN response in vertebrates[END_REF][START_REF] Holland | Molecular characterization of IRF3 and IRF7 in rainbow trout, Oncorhynchus mykiss: functional analysis and transcriptional modulation[END_REF], which is not an ISG in mammals. Although their induction pathways are partly unknown, IFN-independent induction has been observed for some of them. Whether regulatory loops of signaling pathways for type I IFN and ISGs induction are ancestral, or have been shaped independently during fish versus tetrapod evolution, remains to be clarified.

The evolution of teleost fish was marked by an early WGD event, followed by a gene loss phase, and as a consequence, the fish genomes sequenced to date do not contain more genes than humans, but paralogous pairs that arose from this WGD are frequent [START_REF] Howe | The zebrafish reference genome sequence and its relationship to the human genome[END_REF]. To further complicate things, additional WGD episodes occurred in some branches among teleosts-for example, in salmonids-while other fish underwent strong genome contraction, such as the tetraodon/fugu family. Of note, zebrafish has a relatively large genome with many highly expanded gene families, compared to other fish model species [START_REF] Pasquier | Fish "n" TRIMs[END_REF]. Since genes involved in effector mechanisms of immunity tend to diversify to escape subversion by pathogens, one might expect that fish would have retained many ISG duplicates and would possess larger repertoires of ISGs.

In fact, this hypothesis is still difficult to validate, since the diversity of fish ISGs is not fully defined. A few typical ISGs were first identified using primers or probes targeting conserved sequences such as Mx [START_REF] Trobridge | Characterization of a rainbow trout Mx gene[END_REF][START_REF] Trobridge | Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells[END_REF][START_REF] Robertsen | Molecular cloning of double-stranded RNA inducible Mx genes from Atlantic salmon (Salmo salar L.)[END_REF] and genes of the MHC class I presentation pathway [START_REF] Hansen | Induction of the rainbow trout MHC class I pathway during acute IHNV infection[END_REF]. Then, PCR-based approaches for differential display of transcripts (differential display PCR, subtractive suppressive hybridization, etc.) led to the discovery of genes with high induction level; for example, viperin/vig1 and 20 other viral hemorrhagic septicemia virus (VHSV)-induced genes (vig) including isg15 and two chemokines were identified in rainbow trout leukocytes by DDPCR and SSH [START_REF] Boudinot | vig-1, a new fish gene induced by the rhabdovirus glycoprotein, has a virus-induced homologue in humans and shares conserved motifs with the MoaA family[END_REF][START_REF] O'farrell | Survey of transcript expression in rainbow trout leukocytes reveals a major contribution of interferon-responsive genes in the early response to a rhabdovirus infection[END_REF][START_REF] Boudinot | Viral haemorrhagic septicaemia virus induces vig-2, a new interferon-responsive gene in rainbow trout[END_REF]. cd9 and isg15 were found induced by the rhabdovirus infectious hematopoietic necrosis virus (IHNV) in Atlantic salmon with the same methods [START_REF] Fujiki | Cloning and characterization of cDNA clones encoding CD9 from Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Seppola | Sequence and expression analysis of an interferon stimulated gene (ISG15) from Atlantic cod (Gadus morhua L.)[END_REF], which were applied to many fish species. In grass carp (Carassius carassius), subtractive approaches showed that an irf-like [START_REF] Zhang | Molecular cloning and characterization of crucian carp (Carassius auratus L.) interferon regulatory factor 7[END_REF], jak1 and stat1, two Mx [START_REF] Zhang | Differential expression of two Carassius auratus Mx genes in cultured CAB cells induced by grass carp hemorrhage virus and interferon[END_REF], two isg15 [START_REF] Zhang | Differential expression of two Carassius auratus Mx genes in cultured CAB cells induced by grass carp hemorrhage virus and interferon[END_REF][START_REF] Zhang | Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp[END_REF], and a number of genes encoding tetratricopeptide-containing proteins [START_REF] Zhang | Differential expression of two Carassius auratus Mx genes in cultured CAB cells induced by grass carp hemorrhage virus and interferon[END_REF] are up-regulated by the grass carp hemorrhage virus. In Atlantic cod (Gadus morhua), SSH screening after poly(I:C) stimulation identified a number of genes including those encoding ISG15; IRF-1, IRF-7, and IRF-10; MHC class I; VIPERIN/ VIG1; and the ATP-dependent helicase LGP2 [START_REF] Rise | Functional genomic analysis of the response of Atlantic cod (Gadus morhua) spleen to the viral mimic polyriboinosinic polyribocytidylic acid (pIC)[END_REF]. In the sea bass (Dicentrarchus labrax), brain nodavirus-infected tissue was analyzed and C-type lectins, pentraxin, and an anti-inflammatory galectin were found [START_REF] Dios | Suppression subtraction hybridization (SSH) and macroarray techniques reveal differential gene expression profiles in brain of sea bream infected with nodavirus[END_REF][START_REF] Poisa-Beiro | Nodavirus infection of sea bass (Dicentrarchus labrax) induces up-regulation of galectin-1 expression with potential anti-inflammatory activity[END_REF]. A more comprehensive representation of the fish transcriptional response to viral infection came only with genome and EST high-throughput sequencing, opening the way to the microarray technology. Microarray analyses were applied to characterize the response induced by different viruses [START_REF] Purcell | Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus[END_REF][START_REF] Byon | Comparative immune responses in Japanese flounder, Paralichthys olivaceus after vaccination with viral hemorrhagic septicemia virus (VHSV) recombinant glycoprotein and DNA vaccine using a microarray analysis[END_REF][START_REF] Mackenzie | Comparative analysis of the acute response of the trout, O. mykiss, head kidney to in vivo challenge with virulent and attenuated infectious hematopoietic necrosis virus and LPS-induced inflammation[END_REF][START_REF] Workenhe | Infectious salmon anaemia virus (ISAV) isolates induce distinct gene expression responses in the Atlantic salmon (Salmo salar) macrophage/dendritic-like cell line TO, assessed using genomic techniques[END_REF][START_REF] Krasnov | Genomic analysis of the host response to nervous necrosis virus in Atlantic cod (Gadus morhua) brain[END_REF][START_REF] Schiøtz | Transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells[END_REF], IFN inducers [START_REF] Byon | Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination[END_REF][START_REF] Milev-Milovanovic | Identification and expression analyses of poly [I:C]-stimulated genes in channel catfish (Ictalurus punctatus)[END_REF], or recombinant IFN itself [START_REF] Martin | Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/ SGP microarray[END_REF]. These transcriptome analyses from multiple cell types and tissues suggested that a "core" set of 50-100 genes is typically induced [START_REF] Verrier | Early antiviral response and virus-induced genes in fish[END_REF]. To get a more comprehensive repertoire of ISG in a whole fish, we recently characterized the response of the zebrafish larva to the Chikungunya virus (CHIKV), a virus that induces a powerful type I IFN response [START_REF] Briolat | Contrasted innate responses to two viruses in zebrafish: insight into the ancestral repertoire of vertebrate interferon stimulated genes[END_REF]. A set of highly induced ISGs was found, which is also typically retrieved in human [START_REF] Schoggins | A diverse range of gene products are effectors of the type I interferon antiviral response[END_REF]111]: rsad2, CD9, isg12, isg15, ifit and ifi44 family members, stat1, trim25, socs1, irf1, and irf7. This gene set was concordant with the major list of fish ISGs predicted from different tissues of other species (see above, reviewed in Ref. [START_REF] Verrier | Early antiviral response and virus-induced genes in fish[END_REF]). A list of zebrafish orthologues of human ISGs was similar to the repertoire of genes up-regulated by CHIKV infection, which also further confirmed the size of this core set [START_REF] Briolat | Contrasted innate responses to two viruses in zebrafish: insight into the ancestral repertoire of vertebrate interferon stimulated genes[END_REF].

The above-mentioned analysis of the zebrafish orthologues of all human ISGs also revealed some important mammalian ISGs that are almost certainly lacking an orthologue in the zebrafish genome [START_REF] Briolat | Contrasted innate responses to two viruses in zebrafish: insight into the ancestral repertoire of vertebrate interferon stimulated genes[END_REF]. Zebrafish (and apparently all teleosts) lacks the APOBEC3, OAS, IFI16, and CLEC4 families altogether. Among other notable absent genes, one may cite bst2/tetherin; several trim such as trim5, trim22, or pml/trim19; and isg20.

A significant antiviral activity was demonstrated in fish for several of the ISGs. For example, overexpression of a Japanese flounder PKR homologue increased eIF-2 phosphorylation and inhibited the replication of the Scophthalmus maximus rhabdovirus [START_REF] Zhu | Functional domains and the antiviral effect of the double-stranded RNAdependent protein kinase PKR from Paralichthys olivaceus[END_REF]; MX proteins blocked the birnavirus IPNV [START_REF] Larsen | Inhibition of infectious pancreatic necrosis virus replication by atlantic salmon Mx1 protein[END_REF], but not the rhabdovirus IHNV [START_REF] Trobridge | Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells[END_REF]; fish ubiquitin-like ISG15 shares with its mammalian homologues the anchor LRGG motifs and interacts with cellular and viral proteins [START_REF] Røkenes | Atlantic salmon ISG15: Expression and conjugation to cellular proteins in response to interferon, double-stranded RNA and virus infections[END_REF], and an ISGylation-dependent activity of the zebrafish ISG15 was recently demonstrated against different RNA and DNA viruses [START_REF] Langevin | Zebrafish ISG15 exerts a strong anti-viral activity against RNA and DNA viruses and regulates the interferon response[END_REF]. A cytokine-like activity was also reported for the ISG15 secreted form in the tongue sole [START_REF] Wang | Cynoglossus semilaevis ISG15: a secreted cytokine-like protein that stimulates antiviral immune response in a LRGG motifdependent manner[END_REF], as previously for mammals [START_REF] Cunha | In vitro and in vivo secretion of human ISG15, an IFN-induced immunomodulatory cytokine[END_REF].

Altogether, these observations indicate that a number of essential ISGs were already important players of the IFN-mediated antiviral response rather early in the vertebrate history, at least in the common ancestor of tetrapods and fish. It starts to be possible to assess the extent of functional conservation of this core gene set, not only by direct comparison of the functions of individual genes but also using global comparative analyses. For example, some ISGs are typically induced more than others. Do human ISGs and their zebrafish homologues show similar response patterns? Figure 3A shows a tentative correlation of the response of zebrafish larva to CHIKV with the response of human liver to IFNα [START_REF] Sarasin-Filipowicz | Interferon signaling and treatment outcome in chronic hepatitis C[END_REF] and illustrates that orthologues of strongly induced human ISGs tend to be strongly induced by CHIKV infection in zebrafish as well.

Genes involved in immune responses typically show high rates of evolution due to selection pressures exerted by pathogen subversion. Under this rule, ISGs should show a similar trend, and we should observe a negative correlation between ISG sequence similarity in fish and human and their induction level. The relationship between induction rate and sequence similarity/conservation is obviously complex, and these two parameters are not merely correlated (Fig. 3B). However, the global pattern may suggest a loose negative correlation, and outliers such as rsad2/viperin, which are highly conserved and well induced by IFN, constitute interesting exceptions.

Many ISGs are members of gene families, with different evolutionary dynamics of expansion/ diversification during the evolution of tetrapods versus that of fish. Among families containing ISGs, two different patterns were observed: families that differentiated in parallel in tetrapods and fishes from a single common ancestor gene ("young" families) and families that had already diversified in the common ancestor to fishes and mammals ("old" families) [START_REF] Briolat | Contrasted innate responses to two viruses in zebrafish: insight into the ancestral repertoire of vertebrate interferon stimulated genes[END_REF]. Young families (such as MX or IFIT) would likely bind viral components and quickly diversify under strong selection pressure. On the contrary, old, stabilized families typically contain regulatory factors or signal transduction components (i.e., IRFs, STATs, and SOCS) and constitute key molecules in the conserved antiviral machinery.

To illustrate how comparative analysis of human and fish transcriptional responses might suggest important new genes to be targeted in future studies, we will focus on the subset of human ISGs that have a one-to-one orthologue in zebrafish, because they are the easiest to test experimentally, for example, by morpholino knockdown assays. This list includes 178 human genes [START_REF] Briolat | Contrasted innate responses to two viruses in zebrafish: insight into the ancestral repertoire of vertebrate interferon stimulated genes[END_REF]. Strikingly, among these ISGs, 140 (80%) are not annotated as having a potential role in antiviral defense in the current Ensembl GO classification. Some of those genes surely play important, but for the moment overlooked, roles in antiviral responses. Good candidates for further research would be ancestral ISGs, identifiable within this list by having a zebrafish orthologue induced by IFN. At least four genes fulfill this criterion based on the microarray analysis of the response to CHIKV: cmpk2, phf11, upp2, and ftsjd2. The kinase CMPK2 participates in dUTP and dCTP synthesis in mitochondria and may play a role in monocyte differentiation, PHF11 is a positive regulator of Th1-type cytokine gene expression, UPP2 is involved in nucleoside synthesis, and FTSJD2 mediates mRNA cap1 2′-O-ribose methylation to the 5′-cap structure of mRNAs-a feature that, remarkably, distinguishes host mRNAs from some viral mRNAs [START_REF] Daffis | 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members[END_REF]. More genes shall be added to this list in the future as RNA-seq analysis and improved stimulation protocols will yield a more exhaustive list of zebrafish ISGs.

Part 3. IFN-Producing Cells

The current paradigm for type I IFN production in mammals is that all cell types are able to produce IFNβ upon sensing a virus, and in addition, some specialized sentinel cells such as plasmacytoid dendritic cells can produce very high levels of IFNα. The specialized cells have a different array of sensing molecules (e.g., TLR7) and are poised for rapid IFN expression by constitutive expression of some signal-transducing molecules that need to be induced in other cell types (e.g., IRF7). Is the situation similar in fish?

A few studies have addressed the tissue-specific differences in expression of fish type I IFNs and sometimes identified the cell types involved. Zou et al. [START_REF] Zou | Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates[END_REF] found important differences between leukocytes and fibroblasts upon poly(I:C) stimulation in vitro: thus, head kidney cells would express all IFNs tested, while RTG-2 fibroblasts would express the group I IFNs (IFN1 and IFN2) but not the group II IFN (IFN3). Ex vivo analysis of tissues from infected trout suggested a similar picture, with IFN3 being expressed in lymphoid tissue (kidney and spleen) but much less in liver [START_REF] Zou | Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates[END_REF]. In Atlantic salmon, Sun et al. [START_REF] Sun | Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties[END_REF] also found a much more restricted expression of IFN subtype by fibroblast-like TO cells, where only IFNa (a group I IFN) was induced more than twofold, while head kidney leukocytes would also express the group II IFNb and IFNc [START_REF] Sun | Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties[END_REF]. In these cells, polyI:C would induce IFNa and IFNc, while S-27609 (a TLR7 agonist) would preferentially induce IFNb. Similar outcomes were found in vivo at early time points after poly(I:C) or S-27609, but the pattern changed strongly after a few days, likely as a result of complex feedback loops [START_REF] Sun | Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties[END_REF]. More recently, Svingerud et al. published a study that largely confirmed these findings (using R848, a TLR7/8 agonist, instead of S-27609) and added much spatial information, notably by performing in situ hybridization on tissue slices [START_REF] Svingerud | Atlantic salmon type I IFN subtypes show differences in antiviral activity and cell-dependent expression: evidence for high IFNb/IFNc-producing cells in fish lymphoid tissues[END_REF]. Quite remarkably, in all tissues, expression of all tested IFNs was restricted to a minority of cells. IFNa and IFNc were sometimes coexpressed by the same cell in poly(I:C)-injected animals, while IFNb and IFNc could be coexpressed after R848 injection. Cell types that could be identified as expressing IFNs were Fig. 3. Assessment of the conservation of ISGs: comparison of induction levels and sequence similarity between human ISGs and their zebrafish orthologues. (A) Induction levels of human ISGs (liver biopsy cells treated for 4 h with IFNα, from Sarasin-Filipowicz et al. [START_REF] Sarasin-Filipowicz | Interferon signaling and treatment outcome in chronic hepatitis C[END_REF], GEO accession GSE11190) compared with induction levels of their zebrafish orthologues (larvae infected for 48 h with the strong IFN-inducing CHIKV, GEO accession GSE47057). When homologous genes from human and zebrafish were not linked by a one-to-one orthology relationship, they were linked by a colored dotted line and set at the geometric average of the fold changes values of the other species. In these cases, the name of the gene family is indicated in the corresponding color. (B) Level of induction by CHIKV of zebrafish genes orthologous to human ISGs [same data set as for (a)], compared with their degree of similarity with their human orthologues (retrieved from the Ensembl database). endothelial cells and gill pillar cells for IFNa and gill pillar cells for IFNc. No IFNb-expressing cell could be positively identified, but the data suggest that they were distinct from IFNa-expressing cells. IgM-positive B cells did not express any IFN; neither did melanomacrophages [START_REF] Svingerud | Atlantic salmon type I IFN subtypes show differences in antiviral activity and cell-dependent expression: evidence for high IFNb/IFNc-producing cells in fish lymphoid tissues[END_REF].

More recently, this question has been addressed in zebrafish using IFN-reporter transgenes. In larvae, among the four zebrafish ifn genes, only ifnφ1 (a group I ifn) and ifnφ3 (a group II ifn) are considered to play a role, because ifnφ2 is expressed only at the adult stage and ifnφ4 does not seem to exert a significant antiviral effect [START_REF] Aggad | The two groups of zebrafish virusinduced interferons signal via distinct receptors with specific and shared chains[END_REF]. An ifnφ1 reporter transgene has been recently reported [START_REF] Palha | Real-time whole-body visualization of Chikungunya virus infection and host interferon response in zebrafish[END_REF] and analyzed in the context of CHIKV infection, which induces a strong IFN response. The transgene was mainly expressed in two cell populations: neutrophils and hepatocytes-a pattern entirely consistent with expression of the endogenous ifnφ1 gene as seen by in situ hybridization, although the transgene expression was somewhat delayed [START_REF] Palha | Real-time whole-body visualization of Chikungunya virus infection and host interferon response in zebrafish[END_REF]. The pathways inducing ifnφ1 in these two populations are not yet unraveled but are likely to be different since hepatocytes were a target of CHIKV while neutrophils were not infected. A small macrophagelike population also expressed the transgene. Depletion studies demonstrated that neutrophils, but neither hepatocytes nor macrophages, were critical to control the infection. Interestingly, in control, uninfected fish, a small population of neutrophils (10-30 cells/larva) express the transgene at a weak level [START_REF] Palha | Real-time whole-body visualization of Chikungunya virus infection and host interferon response in zebrafish[END_REF]. An IFNφ3 reporter line has also been generated (V. Briolat, N.P., G. Lutfalla, and J.-P.L., unpublished results). The pattern of expression of this transgene during CHIKV infection is very different from that of the ifnφ1 reporter and includes fibroblasts, endothelial cells, hepatocytes, and muscle fibers, all cell types that may be infected by CHIKV; however, expression of the transgene was only observed in virus capsidnegative cells (N.P., unpublished results).

As a general conclusion, fish IFNs generally appear to be expressed by discrete, scattered cell populations with little overlap between IFN subtypes. Some IFNs are expressed in an "IFNβ" pattern, by fibroblasts and other tissue cells that may be direct targets of the viruses, while others are expressed in an "IFNα" fashion by more specialized immune cells. Surprisingly, however, while group II IFNs are those that are preferentially expressed by hematopoietic cells in salmonids, the reverse seems true in zebrafish: group I is preferentially expressed by neutrophils.

There is so far no evidence for a cell type similar to plasmacytoid dendritic cells in fish, but these studies are still in their infancy. Neutrophils seem to play such a role in zebrafish larvae, which came as a surprise. It remains to be tested whether neutrophils are also major IFN-producing cells in adult zebrafish, in other fish species, and possibly during some viral infections in tetrapods.

Part 4. Kinetics of the Different IFN Responses in Fish

Early studies in fish cell lines described a quick and early production of IFN-like activity after viral infection or incubation with UV inactivated viruses [START_REF] Oie | Reovirus type 2: induction of viral resistance and interferon production in fathead minnow cells[END_REF][START_REF] Beasley | Latent infection in marine fish cell tissue cultures[END_REF]. IFN production following a virus infection was also demonstrated in vivo in rainbow trout, with higher amount on day 1 post-VHSV infection and declines to background level by day 14 post-infection [START_REF] De Kinkelin | Interferon production in rainbow trout (Salmo gairdneri) experimentally infected with Egtved virus[END_REF]. In keeping with this, in carp injected with 10 7 pfu of virulent spring viremia of carp virus, the IFN-like activity peaked as early as days 1 and 2, started to decline at day 3, and had disappeared by day 14 [START_REF] De Kinkelin | Interferon synthesis in trout and carp after viral infection[END_REF].

In the 1990s, the kinetics of the antiviral response was studied in further detail using (semi)Q RT-PCR to assess expression of ISG transcripts. After the first fish type I IFN genes were cloned in the 2000s, the kinetics of the IFN mRNA itself could be measured in various infection contexts. Different types of kinetics were obtained, a few of which will be illustrated.

McBeath et al. compared the kinetics of type I IFN in Atlantic salmon after infection by ISAV and IPNV [START_REF] Mcbeath | Expression kinetics of interferon and interferon-induced genes in Atlantic salmon (Salmo salar) following infection with infectious pancreatic necrosis virus and infectious salmon anaemia virus[END_REF]. Type I IFN and Mx expression peaked twice on days 3 and 6 after IPNV infection and declined progressively. This biphasic response might rely on a positive feedback loop depending on IRF induction by the first burst of IFN production as described in mammals [START_REF] Marié | Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7[END_REF]; however, the mechanisms underlying the biphasic salmon IFN response to IPNV remain unknown. In contrast to this kinetics, a later, monophasic type I IFN response occurred after ISAV infection; IFN shortly peaked on day 5 or 6, while Mx peaked on day 6, declined to day 9, and remained expressed until day 30 post-infection. These differences likely reflected that these viruses use different mechanisms for dealing with the host response. Early up-regulation of IFN and ISG like Mx by the IPNV probably contributed to the good survival recorded after this infection. In contrast, high mortality and late response were observed after ISAV infection, which could be due to viral anti-IFN mechanisms [START_REF] García-Rosado | Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity[END_REF]. Transcriptome profiling of the response induced by recombinant IFN in macrophage-like SHK1 cells showed that Mx and other ISGs were induced after 6 h of incubation and peaked at 24 h [START_REF] Martin | Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/ SGP microarray[END_REF], supporting other observations reported for different tissues (e.g., trout kidney leukocytes in Ref. [START_REF] O'farrell | Survey of transcript expression in rainbow trout leukocytes reveals a major contribution of interferon-responsive genes in the early response to a rhabdovirus infection[END_REF]).

However, these studies do not reflect the whole complexity of the type I IFN response since (1) most of the first QPCR and array systems did not take into account the IFN alternative transcripts discovered in zebrafish and in other species; hence, measures of IFN up-regulation integrate both secreted and non-secreted isoforms, which provides a partial view of the kinetics of the effective response; (2) fish genome and EST sequences revealed many type I IFN genes, especially in salmonids; (3) IFNγs may also contribute to the induction of some ISGs [START_REF] Sun | Antiviral activity of salmonid gamma interferon against infectious pancreatic necrosis virus and salmonid alphavirus and its dependency on type I interferon[END_REF].

It is difficult to compare kinetics of IFN gene induction by two different viruses; not only is there a large range of antiviral mechanisms potentially at play (as discussed later), but viral burden (and thus signal) is likely to be different in both cases; comparing induction of different genes in the same context is more informative. For instance, in the zebrafish CHIKV infection model, expression of ifnφ1 was sustained, while ifnφ3 expression was more transient [START_REF] Palha | Real-time whole-body visualization of Chikungunya virus infection and host interferon response in zebrafish[END_REF]. This likely reflects the different pathways (and cell types, as discussed above) involved in their induction, consistently with results of luciferase assays suggesting the variable contribution of IRF3 and/or IRF7 to activate the promoters of the various zebrafish IFNs [START_REF] Sun | Fish MITA serves as a mediator for distinct fish IFN gene activation dependent on IRF3 or IRF7[END_REF].

Part 5. Tissue-Specific Responses

Expression of IFNs is induced upon detection of viruses and is thus expected to be fairly organ specific, depending on the tropism of the particular virus considered. By contrast, since type I IFN receptors are ubiquitously expressed in mammals and IFNs diffuse via the blood, ISGs would be expressed in a more uniform fashion. However, recent findings have shown this idea to be simplistic. For instance, type III IFNs induce the same set of ISGs than type I IFNs, but their receptor is expressed in a tissue-restricted fashion, allowing for targeted induction of ISGs, notably in epithelia exposed to outer environment such as the gut [START_REF] Zhou | Type III interferon (IFN) induces a type I IFN-like response in a restricted subset of cells through signaling pathways involving both the Jak-STAT pathway and the mitogen-activated protein kinases[END_REF]. In addition, even upon systemic type I IFN administration, ISG expression has been found to be highly variable from tissue to tissue [START_REF] Pulverer | Temporal and spatial resolution of type I and III interferon responses in vivo[END_REF]. Do we find a similar situation in fish?

As mentioned above, fish also possess two groups of virus-induced IFNs that signal via two distinct receptors [START_REF] Aggad | The two groups of zebrafish virusinduced interferons signal via distinct receptors with specific and shared chains[END_REF]. Although both groups are phylogenetically related to mammalian type I (rather than type III) IFNs [START_REF] Hamming | Crystal structure of Zebrafish interferons I and II reveals conservation of type I interferon structure in vertebrates[END_REF], it has been proposed that the group I/group II and type I/type III dichotomies may have evolved in a convergent manner in teleosts and tetrapods, respectively [START_REF] Aggad | The two groups of zebrafish virusinduced interferons signal via distinct receptors with specific and shared chains[END_REF]. A potential selective advantage of the dichotomy would be that a response restricted to external tissues may deal with most viruses with few of the side effects associated with a full-blown IFN response, which would be triggered only upon the most severe viral infections. Unfortunately, there are as yet no data published regarding the tissue-specific expression of the receptors for the two groups of IFNs. Both receptors share the CRFB5 chain, which is expressed ubiquitously at a relatively high level, but the weak expression of the specific CRFB1 and CRFB2 chains precluded their detection by whole-mount in situ hybridization in zebrafish embryos [START_REF] Levraud | Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system[END_REF].

We also recently used whole-mount in situ hybridization to establish the expression pattern of four ISGs (isg15, rsad2/viperin, isg12.1, and irf7) in zebrafish larvae, notably in the CHIKV infection model, which results in a very strong endogenous IFN expression [START_REF] Briolat | Contrasted innate responses to two viruses in zebrafish: insight into the ancestral repertoire of vertebrate interferon stimulated genes[END_REF]. Basal levels of expression were below detection level, but upon infection, strongly tissue-dependent induction was observed, with an overall pattern of expression in liver, gut, and blood vessels, with some gene-specific differences (e.g., viperin was comparatively less induced in the gut while isg12.1 was less induced in the liver). A rather similar, if weaker, pattern was observed after IHNV infection [START_REF] Briolat | Contrasted innate responses to two viruses in zebrafish: insight into the ancestral repertoire of vertebrate interferon stimulated genes[END_REF] or after intravenous injection of recombinant zebrafish IFNs (J.-P.L., unpublished results), suggesting that it mostly reflects the differential susceptibility of organs to circulating IFNs.

It is still unclear whether this pattern seen in zebrafish larvae can be generalized, as tissue variability in ISG expression has been addressed in relatively few studies. Lymphoid organs constitute the site for the activation of a proper immune response and, therefore, the majority of the studies present in literature focus their attention on the specific responses activated in those tissues. Responses have also sometimes been analyzed in some tissues for which viruses were known to have a preferred tropism. The following paragraphs focus on such studies.

One of the gateways of viral entry and replication in fish is fin bases, for example, for novirhabdoviruses [START_REF] Harmache | Bioluminescence imaging of live infected salmonids reveals that the fin bases are the major portal of entry for Novirhabdovirus[END_REF]. In response to lethal VHSV infection of Pacific herring (Clupea pallasii), Mx, psmb9, and an MHC class I gene were found to be induced both in the spleen and in the fin bases, with a moderately stronger induction in the spleen attributed to the higher viral burden in this organ [START_REF] Hansen | Induction of anti-viral genes during acute infection with Viral hemorrhagic septicemia virus (VHSV) genogroup IVa in Pacific herring (Clupea pallasii)[END_REF]. Transcriptomic and proteomic studies performed in adult zebrafish during VHSV infection have shown that a number of infection-related genes/proteins are overexpressed in the fins but not in other organs. Among these are complement components, interleukin genes, hmgb1 protein, mst1, and cd36 [START_REF] Encinas | Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach[END_REF]. This does not seem to reflect a typical ISG response, and indeed ifnφ1 transcripts were not identified in this study, possibly because the low temperature required for VHSV replication was suboptimal for induction of a response in zebrafish. Infection of rainbow trout fin bases with VHSV, on the other hand, determines the up-regulation of the chemokines CK10 and CK12, as opposed to those overexpressed in the gills (CK1, CK3, CK9, and CK11). These expression variations may be due to a different permissivity of the tissues (fins or gills) to viral replication [START_REF] Montero | Specific regulation of the chemokine response to viral hemorrhagic septicemia virus at the entry site[END_REF].

Several fish viruses are also known to have a tropism for the heart. Fish alphaviruses and, more recently, members of the Totiviridae family (e.g., piscine myocarditis virus) are associated with cardiac and/or skeletal myopathies. In particular, alphaviruses, such as salmonid alphavirus subtype-1, are capable of causing acute heart lesions with necrotic foci and hypertrophy of the cardiac muscle. Unlike adult fish, smolts can replace damaged cardiomyocytes by cell division and may, therefore, be subjected to a decreased pathogenesis and impact [START_REF] Mcloughlin | Alphavirus infections in salmonids-a review[END_REF]. Recently, the determinants of resistance of two strains of Atlantic salmon to salmonid alphavirus have been investigated, comparing responses in heart, kidney, and gills (a possible port of virus entry). The two strains displayed significantly different basal expressions of ifna1 and ISGs (Mx, viperin, and cxcl10); however, the induction by viral infection was comparable in the three organs [START_REF] Grove | Immune parameters correlating with reduced susceptibility to pancreas disease in experimentally challenged Atlantic salmon (Salmo salar)[END_REF]. Similar results were obtained from Atlantic salmon infected with piscine myocarditis virus [START_REF] Timmerhaus | Transcriptome profiling of immune responses to cardiomyopathy syndrome (CMS) in Atlantic salmon[END_REF].

Several fish viruses also have a preferred tropism for the central nervous system. One of the most serious viral diseases affecting marine fish is represented by nodavirus encephalopathy. The central nervous system and the eye constitute the specific targets for nodavirus replication, leading to mass mortality in larvae and juvenile fish. Numerous studies have, therefore, been conducted to determine the immune responses activated in the brain tissue upon infection, but comparison with other tissues remain scarce. Infection of zebrafish larvae with nervous necrosis virus (NNV), for example, leads to mortality rates higher than 95%. This has been linked to the lack of IFN and Mx expression, not detectable in the larval stage but expressed by infected adults [START_REF] Krasnov | Genomic analysis of the host response to nervous necrosis virus in Atlantic cod (Gadus morhua) brain[END_REF]. A thorough transcriptomic analysis conducted in Atlantic cod (G. morhua) has revealed that NNV infection affects mainly neural processes and their regulation and cellular differentiation (down-regulated genes). Many ISGs were found to be induced in the brain, but expression in other tissues was not reported [START_REF] Krasnov | Genomic analysis of the host response to nervous necrosis virus in Atlantic cod (Gadus morhua) brain[END_REF]. NNV infection in turbot (S. maximus) is followed by overexpression of Mx, irf-1, and tnf-α [START_REF] Montes | Nodavirus encephalopathy in turbot (Scophthalmus maximus): inflammation, nitric oxide production and effect of anti-inflammatory compounds[END_REF]. Finally, in European sea bass (D. labrax), two different x genes (MxA and MxB) were differentially expressed during NNV infection. While MxA is highly up-regulated in the brain, MxB expression does not differ substantially from controls, thereby suggesting that the former is the predominant isoform and that MxB may play a different and independent functional role [START_REF] Novel | Two Mx genes identified in European sea bass (Dicentrarchus labrax) respond differently to VNNV infection[END_REF].

Part 6. Subversion Mechanisms by Viruses in Fish

The complexity of antiviral signaling pathways reflects the dynamic interactions between viruses and their hosts and has been shaped by the highly diverse strategies developed by these pathogens to evade antiviral immunity. In mammals, a vast number of strategies have been discovered, targeting immunity (pattern recognition receptors, IFN signaling, MHC class I presentation, cytokine or chemokine networks, etc.) as well as basic mechanisms of virus-host interactions (autophagy, cell cycle, protein synthesis, etc.).

Such mechanisms are certainly used by fish viruses as well, but remain poorly described. Subversion of host immune response has been mainly studied for novirhabdoviruses, birnaviruses, and orthomyxoviruses.

Novirhabdoviruses are negative-sense singlestranded RNA viruses infecting fishes. They have a small genome encoding four structural proteins (N, P, M, and G) plus a polymerase (L), like other rhabdoviruses, and one specific nonstructural protein (NV), which is a good candidate for subversion of immune pathways. Recombinant IHN and VHS viruses lacking NV were able to replicate in cell culture, although the growth of the IHNV-ΔNV was severely impaired [START_REF] Biacchesi | Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes[END_REF][START_REF] Thoulouze | Essential role of the NV protein of Novirhabdovirus for pathogenicity in rainbow trout[END_REF][START_REF] Ammayappan | A reverse genetics system for the Great Lakes strain of viral hemorrhagic septicemia virus: the NV gene is required for pathogenicity[END_REF]. The importance of NV protein for pathogenicity was also strongly suggested by in vivo challenges with mutant viruses that caused only 20% mortality, whereas the wild-type control virus causes 100% mortality [START_REF] Biacchesi | Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes[END_REF][START_REF] Thoulouze | Essential role of the NV protein of Novirhabdovirus for pathogenicity in rainbow trout[END_REF][START_REF] Ammayappan | A reverse genetics system for the Great Lakes strain of viral hemorrhagic septicemia virus: the NV gene is required for pathogenicity[END_REF]. Although the sequence of the NV protein is not highly similar between novirhabdoviruses, the attenuated phenotype of VHSV-ΔNV can be rescued by re-introduction of NV from IHNV and vice versa [START_REF] Thoulouze | Essential role of the NV protein of Novirhabdovirus for pathogenicity in rainbow trout[END_REF][START_REF] Ammayappan | Nonvirion protein of novirhabdovirus suppresses apoptosis at the early stage of virus infection[END_REF], suggesting that the function of NV during infection is conserved. In fact, cells infected by NV-deletion mutants express higher levels of type I IFN transcripts, suggesting that NV is used to evade the innate antiviral immune response [START_REF] Choi | A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth[END_REF]. Moreover, growth of IHNV-ΔNV was inhibited by poly(I:C) treatment at 24 h post-infection, while the wild-type virus was not blocked. The overexpression of VHSV NV protein also reduced the TNFα-mediated activation of NFκB, which likely contributes to its impact on the innate response [START_REF] Kim | The role of viral hemorrhagic septicemia virus (VHSV) NV gene in TNF-alpha-and VHSV infectionmediated NF-kappaB activation[END_REF].

"Multitask" properties are known for M and P proteins of prototypical rhabdoviruses infecting higher vertebrates, rabies virus (RV), and vesicular stomatitis virus (VSV) [START_REF] Rieder | Rhabdovirus evasion of the interferon system[END_REF]. RV was shown to diminish IFNβ induction through the viral protein P, which blocked IRF3 phosphorylation [START_REF] Brzózka | Identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein P interferes with phosphorylation of interferon regulatory factor[END_REF]. The P protein of RV also inhibited IFN downstream signaling by blocking the nuclear import of STAT1 [START_REF] Vidy | Rabies virus P protein interacts with STAT1 and inhibits interferon signal transduction pathways[END_REF] and has an impact on viral transcription and nucleocapsid formation. In fish, such mechanisms have not been reported yet, but the P protein of IHNV (as well as NV) is targeted by ISG15, which may represent a cell countermeasure [START_REF] Langevin | Zebrafish ISG15 exerts a strong anti-viral activity against RNA and DNA viruses and regulates the interferon response[END_REF]. Indeed, overexpression of ISG15 in EPC cells is sufficient to trigger antiviral activity against novirhabdoviruses (IHNV, VHSV), birnavirus (IPNV), or iridovirus (EHNV). ISGylation, which targets cellular proteins such as TRIM25 and viral proteins such as the P and NV of IHNV, is required for viral inhibition: the ISG15 LRAA mutant (incapable of functional ISGylation) does not afford any protection. Subversion of IFN induction has also been demonstrated for fish birnaviruses and orthomyxoviruses. The proteins VP4 and VP5 of the birnavirus IPNV had antagonistic properties towards an IFN reporter [START_REF] Skjesol | The interplay between infectious pancreatic necrosis virus (IPNV) and the IFN system: IFN signaling is inhibited by IPNV infection[END_REF]; however, in vivo comparison of IPNV field isolates with different levels of pathogenicity did not clearly confirm the importance of an intact VP5 protein for virulence [START_REF] Skjesol | IPNV with high and low virulence: host immune responses and viral mutations during infection[END_REF]. Similarly, two ISAV proteins encoded by the genomic segments 7 and 8-respectively named s7ORF1 and s8ORF2are involved in the modulation of the IFN signaling [START_REF] García-Rosado | Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity[END_REF][START_REF] Mcbeath | Identification of an interferon antagonist protein encoded by segment 7 of infectious salmon anaemia virus[END_REF]. While s7ORF1 expression is restricted to the cytoplasm [START_REF] Mcbeath | Identification of an interferon antagonist protein encoded by segment 7 of infectious salmon anaemia virus[END_REF], s8ORF2 possesses two NLS signals responsible for nuclear expression and binds both dsRNA and polyA RNA [START_REF] García-Rosado | Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity[END_REF]. The IFN antagonist activity of s7ORF1 was shown by Mx-Luc reporter assay or RT QPCR on Mx and IFN upon poly(I:C) treatment [START_REF] Mcbeath | Identification of an interferon antagonist protein encoded by segment 7 of infectious salmon anaemia virus[END_REF]. Another study determined that s7ORF1 and s8ORF2 expression down-regulates the activity of a type I IFN promoter upon poly(I:C) exposure [START_REF] García-Rosado | Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity[END_REF].

Large DNA viruses often possess genes blocking IFN pathways or inhibiting ISG function. For example, the ranavirus RCV-Z (Rana catesbeiana virus Z), a pathogen of fish and frogs, circumvents host-induced transcriptional shutoff and apoptosis by expressing a pseudosubstrate for PKR [START_REF] Rothenburg | Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR[END_REF]. Other fish iridoviruses and herpesviruses can also possess such "mimickry" genes: for example, the koi herpesvirus encodes an IL-10 homologue [START_REF] Sunarto | Koi herpesvirus encodes and expresses a functional interleukin-10[END_REF], the Singapore grouper iridovirus encodes IgSF members, and another fish iridovirus encodes a B7-like sequence [START_REF] Hansen | The B7 family of immunoregulatory receptors: a comparative and evolutionary perspective[END_REF].

Viruses also dysregulate a number of basic cellular functions, which they use for their own replication and to block intrinsic antiviral mechanisms. For instance, IHNV has an acute life cycle during which it causes global blockage of cellular transcription, very similarly to the well-studied VSV [START_REF] Chiou | Infectious hematopoietic necrosis virus matrix protein inhibits hostdirected gene expression and induces morphological changes of apoptosis in cell cultures[END_REF][START_REF] Black | Vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo[END_REF]. The M protein of VSV, in addition to repressing cellular transcription, was shown to inhibit nuclear trafficking of RNA and proteins, thereby also inhibiting antiviral responses [START_REF] Her | Inhibition of Ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus[END_REF]. Both VSV and IHNV elicit cell rounding, probably by interfering with cytoskeletal dynamics [START_REF] Chiou | Infectious hematopoietic necrosis virus matrix protein inhibits hostdirected gene expression and induces morphological changes of apoptosis in cell cultures[END_REF][START_REF] Blondel | Role of matrix protein in cytopathogenesis of vesicular stomatitis virus[END_REF]. Shutoff of basic cellular machinery eventually leads to apoptosis. Programmed cell death being also one of the host's antiviral strategies, many viruses developed strategies to delay apoptosis and complete their infection cycles. In fish, VHSV was able to block experimentally induced apoptosis in EPC cells in an NV-dependent manner [START_REF] Ammayappan | Nonvirion protein of novirhabdovirus suppresses apoptosis at the early stage of virus infection[END_REF].

Conclusion

Antiviral immunity has been studied only in a few fish species, either aquaculture fishes or model species. Fish are vertebrates and share with humans and mice most of the key antiviral pathways.

However, fishes had a long and complex genome history and developed a specific adaptation to the aquatic environment (and to its pathogens). Hence, the fish antiviral immunity represents an alternative version of what could evolve upon highly selective pressures of host-virus interactions, from the ancestral system present in the early vertebrates. Comparison of mammalian and fish innate antiviral mechanisms will be certainly beneficial to distinguish the core system, which is resilient to the subversive selective pressures exerted by the viral world, from the specialized systems that emerged during the evolution of each branch in response to particular viral strategies. In addition, the imaging possibilities offered by model fish species such as the zebrafish will be instrumental, in the future, to unravel the spatiotemporal dynamics of these core antiviral responses shared by all vertebrates.

Figure

  Figure I1.1 p.6 Figure I1.2 p.6 Figure I1.3 p.7 Figure I1.4 p.9 Figure I1.5 p.11 Figure I1.6 p.13 Figure I2.1 p.18 Figure I2.2 p.19 Figure I2.3 p.20 Figure I2.4 p.21 Figure I2.5 p.23 Figure I3.1 p.30 Figure I3.2 p.32 Figure I3.3 p.34 Figure I3.4 p.35 Figure I3.5 p.37 Figure I3.5 p.38 Figure 1 p.56 Figure 2 p.58 Figure 3 p.60 Figure 4 p.62 Figure 5 p.63 Figure 6 p.65 Figure S1 p.66 Figure S2 p.67 Figure S3 p.68 Figure S4 p.68 Figure R2.1 p.84 Figure R2.2 p.85

  single-stranded RNA viruses belonging to various families. The DNA African Swine Fever virus represents the only exception. All other neurotropic arboviruses either belong to the Togaviridae, Flaviviridae (West Nile virus (WNV), Dengue virus (DENV)), Bunyaviridae (Hantavirus, Crimean-Congo Hemorrhagic Fever virus, Rift Valley Fever virus), Rhabdoviridae (Rabies virus (RV)),

Figure I1. 1 .

 1 Figure I1.1. Vertebrate host and vector transmission cycles. (A) Enzootic, the natural transmission of virus between wild animals and primary vectors. (B) Epizootic, the transmission of virus between domestic animals and primary vectors. (C) Urban, the virus cycles between humans and insect vectors, due to high level of viremia. Adapted from Go et al. (2014).

Figure I1. 3 .

 3 Figure I1.3. Incidence of human West Nile virus neuroinvasive disease by State as of November 2015 -United States. Ranging from 0.01 to greater than 1.00 per 100,000 population. Source: CDC.

Figure I1. 4 .

 4 Figure I1.4. Primary viremia, after injection of La Crosse virus in weanling mice. Viremia appears as a sharp peak limited to the first 12 hrs. Even if transient, viremia is sufficient to initiate a lethal encephalitis after delivery of the virus to the target organ. Adapted from Pekosz et al. (1995).

1. 3 . 1 .

 31 Infection of Brain Microvascular Endothelium Several viruses are known to enter the CNS by infecting Brain Microvascular Endothelial Cells (BMVECs) of the BBB. Subsequently, they induce disruption of BBB integrity and unrestrained migration of cells of the immune system. Under physiological conditions, the BBB is principally constituted by an interface of glial and vascular cells tightly joined to restrict the movement of solutes and cells from the bloodstream to the brain parenchyma. The endothelial cells lining the microvasculature within the CNS are adjoined by tight and adherens junctions, and receive support from pericytes and astrocytes for their homeostasis. Together with perivascular macrophages, microglia, and neurons these structures constitute the neurovascular unit (Figure I1.5). Upon infection of BMVECs, several neurotropic viruses have been shown to alter junction expression and functionality as well as to induce expression of inflammatory mediators by other BBB cell types. Examples of these viruses include DNA viruses, such as John Cunningham virus (JCV), a human

Figure I1. 5 .

 5 Figure I1.5. Cellular components of the BBB. The barrier is formed by capillary endothelial cells, surrounded by basal lamina and astrocytic perivascular endfeet. Astrocytes provide the link to neurons. Adapted from McGavern and Kang (2011).

  of infection in the CNS, or viral neurotropism, is a direct consequence of cellular susceptibility and viral dissemination, which in turn is preceded by systemic spread (by viremia or the neural route). Upon entry in the CNS, cell-to-cell transmission ensues and the virus starts spreading. Neurotropic viruses may use more than one mechanism, although they generally exhibit cell tropism, by infecting one cell type more readily than others. Viral progression can develop in two prototypical ways: 1. Sequential cellular infection, via axonal transport of virions from one neuron to the connected one 2. Transit via periaxonal accessory cells that surround neurons Axonal transport is by far the best characterized. In this case, viruses employ cellular transport systems to move along axonal projections in an anterograde and/or retrograde manner (Figure I1.6).

Figure I1. 6 .

 6 Figure I1.6. Viral axonal spread. (A) Anterograde spread involves movement of the virus from the soma to the synapse and infection of neighboring cells. (B') Trans-synaptic spread allows movement from the synapse to the cell body (e.g. RV, PRV). (B'') Microfusions between neighboring cells allow retrograde spreading of measles virus. Adapted from McGavern and Kang (2011).

  cells are normally protected from the dangerous side-effects of inflammatory and immune responses. Infected cells are normally spared from immunological effector mechanisms if they are located in the brain. This in turn might contribute to virus persistence the brain. Immune cell entry restriction exerted by the BBB and limited expression of Major Histocompatibility Complex 2 (MHC) class I molecules from neurons -that renders them unable to activate virus-specific cytolytic T lymphocytes -also account for virus persistence in this organ. The brain must therefore employ virus-specific immune mechanisms for the clearance of the pathogen that do not involve cytolytic elimination of the infected cell and consequent CNS damage. While DNA viruses undergo replication in the nucleus and have the capacity to remain latent, most RNA viruses (such as neurotropic arboviruses) replicate in the cytoplasm and do not establish latency 3 . Unlike DNA viruses, they are therefore more susceptible to immune control mechanisms that are readily activated in the CNS upon detection of infected cells. Production of type I Interferon (IFN) cytokines constitutes the first line of defense against viral infections and it is vital for the survival of the infected host. Mice lacking the IFN receptors (IFNAR -/-) show in fact a greater susceptibility to fatal disease, including to infections of the CNS (Muller

  IFN production is fundamental to counteract viral replication and spread as well as for the subsequent induction of an adaptive immune response. The activation of the IFN signaling pathway in fact contributes to the upregulation of MHC class I molecules on the surface of microglial cells and of adhesion molecules by BMVECs. Subsequently, several induced cytokines (e.g. Interleukin-1 (IL-1),,, Tumor Necrosis Factor-α (TNF-α))

Figure I2. 1 .

 1 Figure I2.1. Phylogenetic tree of Alphaviruses from complete structural polyprotein amino acid sequences. From Powers et al. (2011).

  are composed of a host cell-derived lipid bilayer, two membrane-associated glycoproteins (E1 and E2) bound with E3, and a nucleocapsid enclosing the viral genome. 240 monomers of capsid protein are arranged together to form a T = 4 icosahedral structure, studded with 80 heterotrimeric glycoprotein spikes.

Figure I2. 2 .

 2 Figure I2.2. Alphavirus genome and virion structure. (A) The genome is single-stranded positive-sense RNA, encoding two Open Reading Frames. (B) Left, surface shaded view of SINV at 9Å resolution. Trimeric spikes are seen in blue, the small portions of the lipid bilayer are seen in green. Right, central cross-section of the virus showing in blue the glycoproteins, in turquoise the envelope, in green the lipid bilayer, in yellow the capsid, and the RNA region in red. Adapted from Jose et al. (2009).

Figure I2. 3 .

 3 Figure I2.3. Alphavirus life cycle.Upon receptor binding and clathrin-mediated endocytosis, the viral particle fuses with the endosomal membrane. The resulting nucleocapsid is transported to the cytoplasm, where the RNA is released after disassembly. Genomic RNA is used both for translation of proteins from the genomic and subgenomic RNA, as well as for transcription of nascent RNA. Lastly, the structural proteins are translated and encapsidate nascent genomic RNA before budding and release. Adapted from[START_REF] Leung | Replication of Alphaviruses: a review on the entry process of Alphaviruses into cells[END_REF].

  positive-strand genomic and subgenomic RNAs. Except for E2-E3, cleavage of the sPs occurs simultaneously with their translation. The C protein subsequently associates with newly synthesized RNA genomes. The E1/(E2+E3) complexes are then transported from the endoplasmic reticulum; in the Golgi, furin cleaves the E2-E3 bond. At the plasma membrane, the mature E1-2-3 heterotrimers interact with the C protein and form an envelope around nucleocapsid-like particles. Thus, upon release from the infected cell, virions have acquired a membrane bilayer from the host cell plasma membrane(Leung et al., 2011) (Figure I2.4).

Figure I2. 4 .

 4 Figure I2.4. Schematic diagram of Alphavirus genome replication. Non structural proteins are translated and processed first. Later, after transcription of the subgenomic RNA, the structural proteins can be translated and processed. Adapted from Ryman and Klimstra (2008).

  but only half of them are pathogenic in humans. SINV strains have been grouped into five genotypes, based on E2 sequence divergence (Figure I2.5).

Figure I2. 5 .

 5 Figure I2.5. Geography of SINV genotypes (I-V), and suggested route of ancient SINV dispersal from South America to East Asia. Other Aplhaviruses included: Aura, Aura virus; WEEV, Western Equine Encephalitis virus; HJV, Highland J virus; BCV, Buggy Creek virus; FM, Fort Morgan virus. From Lundström and Pfeffer (2010).

Figure I3. 1 .

 1 Figure I3.1. Schematic of zebrafish life stages. Adapted from Levraud et al. (2014).

Figure I3. 2 .

 2 Figure I3.2. (Top) Lateral and (bottom) dorsal view of the adult zebrafish brain. CC, crista cerebellaris; CCe, corpus cerebelli; Ctec, commissura tecti; EG, eminentia granularis; Ha, habenula; IL, inferior lobe of hypothalamus; LL, lateral line nerves; LVII, facial lobe; LX, vagal lobe; MO, medulla oblongata; MS, medulla spinalis; OB, olfactory bulb; PG, preglomerular area; Pit, pituitary; PSp, parvocellular superficial pretectal nucleus; Tel, telencephalon; TeO, tectum opticum; TH, tuberal hypothalamus; TLa, torus lateralis; I, olfactory nerve; II, optic nerve; IV, trochlear nerve; V, trigeminal nerve; VII, facial nerve; VIII, octaval nerve; X, vagal nerve. From Roberts and Ellis (2001).

Figure I3. 3 .

 3 Figure I3.3. Schematic representation of (A) neural crest development and (B) primary neurulation in zebrafish. In (B), mesodermal nuclei are in red, and neural nuclei in yellow. Adapted from Gammill and Bronner-Fraser (2003), and Araya et al. (2014).

  that allows for the stable adoption of a neuronal fate. In the spinal cord, motor neurons occupy central and ventrolateral positions, interneurons are found at intermediate ones, and sensory neurons develop dorsally(Figure I3.5). Somatosensory neural circuits in the developing hindbrain are constituted by different trigeminal sensory neurons. These are groups of ~60 neurons, all extending a single axon along the lateral line of the hindbrain and spinal cord. Later they form numerous branches that innervate nuclei along the anterio-posterior axis of the hindbrain and spinal cord[START_REF] Pan | Robo2 determines subtype-specific axonal projections of trigeminal sensory neurons[END_REF].

Figure I3. 4 .

 4 Figure I3.4. Organization of the neural tube of a 24 hpf zebrafish embryo. Left panel, schematic cross-section, indicating: non-neural ectoderm, RP, roofplate; dorsal, inter-and motor neurons; FP, floorplate; notochord. Right panel, lateral view of a zebrafish embryo in two planes of focus. From Blader and Strähle (2000).

Figure I3. 5 .

 5 Figure I3.5. Schematic representation of upstream IFN signaling pathways in zebrafish. Adapted from Langevin et al. (2013).

Figure I3. 6 .

 6 Figure I3.6. Schematic representation of virus-induced IFNs and their receptors in zebrafish. Adapted from Aggad et al. (2009).

  use of zebrafish to study viral infections. The groups within the Units "Virology and Molecular Immunology" at INRA and "Macrophages and Development of Immunity" at Pasteur Institute led by Pierre Boudinot and Jean-Pierre Levraud respectively, have been able to follow the progression of the infection of a lethal fish rhabdovirus(Ludwig et al., 2011), and more recently of the human Alphavirus related to SINV -Chikungunya virus (CHIKV)(Palha et al., 2013).Moreover, they have developed various transgenic zebrafish lines to follow antiviral responses, such as the ifnφ1:mCherry that labels the main cell populations producing type I IFNs during the infection, and more recently the MxA:mCherry, that labels IFN-responding cells. It has thus been possible to image in real time the progression of the infection in the host, at cellular resolution in the whole body, while simultaneously imaging the host response to the virus.

Figure 1 .

 1 Figure 1. SINV replicates in zebrafish larvae and exhibits a broad organ tropism. (A) Scheme of a 72 hours post-fertilization (hpf) larva, showing the sites of injection: iv, intravenously in the dorsal aorta or in the caudal vein; ic, intracerebrally in the optic tectum; eye, in the retina. (B) Virus titers in iv infected zebrafish larvae. Data represent mean ± s.e.m. of 5 larvae per time-point, from 2 experiments pooled. Dotted line: treshold level from uninfected control larvae. (C) Survival curves of control uninfected (No V) and iv infected zebrafish larvae (SINV). Data pooled from 5 independent experiments. N = 12 larvae per group. (D-E) Superposition of transmitted light and GFP fluorescence. (D) Confocal in vivo imaging of control uninfected (No V) and iv infected (SINV) larvae at 1 day post-infection (dpi). H, heart; L, liver; Y, yolk; white arrowhead: infection in the left pectoral muscle. (E) Confocal in vivo imaging of 3 infected larvae at 1 dpi, illustrating the pattern variability. (E', E'') Lateral views; (E''') Dorsal view; note that E' is a magnification of D''. White arrowheads point to sites of CNS infection. Scalebars: 50 µm. See also Figure S1. In this and all following figures, anterior to left, dorsal to top for lateral views.

Figure 2 .

 2 Figure 2. SINV is neurotropic in zebrafish larvae. (A'-A'''') Confocal in vivo imaging of the same infected larva from 1 to 4 dpi. Blue arrowheads: infected peripheral cells; white arrowheads: infected cells in the CNS. (B, C) Confocal images of whole-mount immunohistochemistry (WIHC) processed SINV-infected larvae, GFP staining (SINV-infected cells) in green, acetylated tubulin (axons) in red. (B) Dorsal view, with inset showing a wider field of the head; dotted square corresponds to the area imaged at higher magnification. (C) Lateral view. White arrowhead: neurons of the trigeminal ganglion. Blue arrowhead: axon from a trigeminal ganglion neuron reaching the optic tectum. (D) Quantification of appearance of infected cells in the periphery and in the CNS, from in vivo observation of HuC:G/U:RFP larvae, infected iv or ic. N = 12-24 from 2 independent experiments pooled. In vivo confocal imaging at (E') 1 dpi and (E'') 2 dpi of a larva infected ic. White arrowheads: CNS infected cells; yellow arrowhead: spinal cord infection; blue arrowhead: infected cells in the eye. Scalebars in (A): 100 µm, in (B, C, E): 50 µm. See also Figure S2.

Figure 3 .

 3 Figure 3. SINV does not infect the brain microvascular endothelium. Confocal imaging of (A, B) fixed and (C) in vivo fli:G/U:RFP larvae at 2 dpi. (A) No V. (B) IHNV-infected larva. (C) SINV-infected larva. (A', B', C') RFP staining (vasculature) in red. (A'', B'', C'') Merge with transmitted light, vasculature in red, virus in green. (A''') No V in green. (B''') G protein of IHNV in green. (C''') SINV-infected cells in green. Scalebars: 50 µm. (D) Confocal imaging from a Clarity-treated fli:G/U:RFP SINV-infected larva at 3 dpi. Scalebar: 250 µm. See also Figure S3.

Figure 4 .

 4 Figure 4. Macrophages are not targeted by SINV. (A) WIHC of SINV-infected mpeg:G/U:Nfsb-mCherry larva at 2 dpi. (A') Representative zebrafish head, with inset showing imaged area. (A'') Macrophages in red, SINV-infected cells in green, cell nuclei in blue (DAPI). (A''') SINV-infected cells. (A'''') Macrophages. Scalebars: 10 µm. (B, C) mpeg:G/U:Nfsb-mCherry were treated with DMSO (control) or Metronidazole (to deplete macrophages), before injection with SINV. (B) Survival curves and (C) Percentage of brain infected larvae in DMSO-treated and Metronidazole-treated larvae. N = 24 from 2 independent experiments pooled. ns -not significant (Log-rank test). See also Figure S4.

Figure 5 .

 5 Figure 5. SINV is transported to the CNS via peripheral neurons. (A) Infection of muscle cells and spinal cord neurons in the tail region. WIHC processed iv-infected larva at 2 dpi. Lateral view, with inset depicting the imaged area. Dotted lines: dorsal and ventral limits of the spinal cord; plain lines: limits of fins; blue arrow: muscle fiber; blue arrowhead: peripheral neuron; white arrowhead: neuron inside the spinal cord. (B) In vivo confocal imaging of SINV-infected larva at 2 dpi, upon injection of the virus in the left eye. Dorsal view, with inset showing a scheme of the projection of the retinal neurons to the optic tectum. White arrowhead: infected cell body in the optic tectum; yellow arrowhead: infected cell in the eye. Probable inoculum leakage indicated by blue arrowhead. Overlay of transmitted light (grayscale) and GFP fluorescence (green). Scalebars: 50 µm.

Figure R1. 6 .

 6 Figure R1.6. A protective IFN-mediated immune response is induced upon SINV-infection. (A-C) Expression of zebrafish type I IFNs (A) ifnφ1, (B) ifnφ3, and (C) IFN-stimulated gene (A-C) Expression of zebrafish type I IFNs (A) ifnφ1, (B) ifnφ3, and (C) IFN-stimulated gene rsad2/viperin. qRT-PCR, mean ± s.e.m. of 3 pools of 9 larvae from 1 representative experiment. (D) In vivo distribution of IFN-producing cells upon infection of ifnφ1:mCherry transgenic larvae at 3 dpi. (D') No V and (D'') SINV-infected larva, iv. White arrowheads: IFN-expressing cells close to infection foci; blue circle: liver; Y: the left part of the yolk was not imaged to reduce acquisition time and results in an incomplete pattern of infection. (E) Survival curves, (F) Disease score, and (E) Percentage of brain infected larvae in Control and IFN-R morphants infected larvae. (E, F) N = 36 from 3 independent experiments pooled. (G) N = 24 from 2 independent experiments pooled. ***P<0.001, *P<0.1, ns -not significant [(E, G) Log-rank; (F) two-tailed unpaired t-test]. (H, I) SINV infection pattern in (H) Control and (I) IFN-R morphants at 2 dpi. Confocal imaging of WIHC processed larvae, GFP in green, acetylated tubulin in red. (D, H, I) Scalebars: 50 µm.

Figure S2 .

 S2 Figure S2. SINV infection in iv and ic injected larvae. Related to Figure 2. (A) Confocal image of WIHC processed SINV-infected larva. GFP staining (SINV-infected cells) in green, acetylated tubulin (axons) in red. Magnification from Figure 2C. White arrowhead: neurons of the trigeminal ganglion. Blue arrowheads: axon from a trigeminal ganglion neuron reaching the optic tectum. (B) Schematic representation of brain areas most commonly infected upon SINV inoculation. Dorsal view of the general organization of the zebrafish brain. OB, olfactory bulb; TeO, optic tectum; CCe, corpus cerebelli; Me, medulla oblongata. (C'-C'''') Confocal images of WIHC processed SINV-infected larvae at 2 dpi, showing infection foci in different areas of the brain. GFP staining (SINV-infected cells) in green, acetylated tubulin (axons) in red. (C', C'') Representative iv-injected larvae, showing infection foci in different areas of the brain. (C''', C'''') Representative ic-injected larvae, showing widespread infection in the brain. (D, E) Effects on (D) Survival and (E) Viral replication upon iv or ic SINV inoculation. (D) Data pooled from 5 independent experiments. N = 12 per group. ***P<0.001 (Log-rank test). (E) Virus titers in iv and ic infected zebrafish larvae. Data represent mean ± s.e.m. of 5 larvae per time-point, from 2 experiments pooled. Dotted line: treshold level from uninfected control larvae.

  The genomes of the two strains differ by 16 mutations, 5 of which encode aminoacid changes (1 in the 5' UTR, 3 in the ORF encoding NSP2 and NSP3, and 2 in the ORF encoding E2) (FigureR2.1).

Figure R2. 1

 1 Figure R2.1 Schematic representation of SINV1-GFP and SINV2-mCherry genome organization. Amino acid changes between SINV1-GFP and SINV2-mCherry sequences are indicated in red.

Figure R2. 2 .

 2 Figure R2.2. Comparison between the two SINV strains, in terms of viral burden, induced host response at 24 hpi, and mortality rate. Left panel, qRT-PCR of SINV E1; Middle panel, qRT-PCR of IFNφ1. Mean + s.e.m. of 3 larvae from 1 representative experiment. ***P<0.001, *P<0.1; Right panel, survival of infected larvae. Data pooled from 5 independent experiments. N = 12 per group. ***P<0.001 (Log-rank test).

  6 TCID 50 /mL) showed increased replication of SINV1-GFP, probably helped by IFN expression shutoff induced by SINV2-mCherry (FigureR2.5).

Figure R2. 3 .

 3 Figure R2.3. Comparison between the effects of SINV2-mCherry and IFN-R knock-down. (A) SINV2-mCherry infected larva. Inset: virus in red, acetylated tubulin in green, with dotted line showing wider panel; high magnification panel: virus in grey, acetylated tubulin in red (pseudocolors). (B) IFN-R morphant infected with SINV1-GFP. Inset: virus in green, acetylated tubulin in red, with dotted line showing wider panel; high magnification panel: virus in grey, acetylated tubulin in red (pseudocolors). Blue arrowheads indicate areas of extensive infection. Scalebars: 25 µm.

Figure R2. 4 .

 4 Figure R2.4. Comparison between SINV1-GFP and SINV2-mCherry. (A) SINV1-GFP in green, acetylated tubulin in red. (B) SINV2-mCherry in red, acetylated tubulin in green. White arrowheads indicate common areas of infection (neurons of the trigeminal ganglion). Scalebars: 50 µm.

Figure R2. 5 .

 5 Figure R2.5. In vivo confocal imaging of a larva co-infected with SINV1-GFP and SINV2-mCherry. SINV1-GFP in green, SINV2-mCherry in red. White arrowhead indicates extensive infection of SINV1-GFP only; blue arrowheads indicate areas of co-infection. Scalebar: 50 µm.

Figure R2. 6 .

 6 Figure R2.6. Progressive infection and IFN-producing cells recruitment in the CNS, upon SINV inoculation. In vivo confocal microscopy of an infected ifnφ1:mCherry larva at (A') 1 dpi, (A'') 2 dpi, and (A''') 3 dpi. Virus in green, IFN-producing leukocytes in red. White arrowheads indicate areas of progressive CNS infection. Scalebars: 50 µm.

Figure R2. 7 .

 7 Figure R2.7. IFN expression in the CNS, upon SINV infection. (A-B''') In vivo confocal imaging of an infected ifnφ1:mCherry larva, at 2 dpi. Virus in green, IFN-producing leukocytes in red, transmitted light in grey. (A') Dorsal view of the head, with dotted line showing high magnification in (B'-B'''). (B') Merge. (B'') SINV-infected neurons. (B''') IFN-expressing cells. Blue arrowheads indicate IFN-expressing SINV-infected neurons, white arrowheads indicate IFN-expressing neutrophils. Scalebars: 25 µm.

Figure R2. 8 .

 8 Figure R2.8. State of microglia, upon SINV infection. Confocal microscopy of IHC-processed (A') No V and (A'') SINV-infected larvae, at 2 dpi. GFP in green, microglia in purple. White arrowheads indicate ramified-shaped microglia, red arrowheads indicate rounded-shaped microglia. Scalebars: 100 µm.

Figure R2. 9 .

 9 Figure R2.9. Image analysis performed with Matlab®. (A) Raw data. (B) Detection of cells based on fluorescence intensity. Red neutrophils are circled in yellow, green infected cells are circled in purple. (A-B) Yellow arrow indicates a GFP-high cell (included in the quantification), blue arrowhead indicates a low-GFP cell (erroneously excluded from the quantification). (C) Quantification of the number of neutrophils and infected cells over time, based on the analysis performed automatically by the algorithm.

  Cells are then plated on pre-coated coverslips and cultured in a modified version of Minimum Essential Medium at 24°C for at least one week. To determine the cell composition of our culture, we established primary cell cultures from transgenic lines expressing the GFP or the mCherry fluorescent proteins in neuronal or in glial cells, respectively (Figure R3.1).

Figure R3. 1 .

 1 Figure R3.1. Zebrafish primary neural cell cultures. In vivo fluorescence microscopy of (A) Huc:G/U:mCherry-derived neurons and (B) Gfap:eGFP-derived glial cells, 1 day post-plating (dpp). Scalebars: 20 µm.

  The experiments performed in vitro by our collaborator Bryan Mounce showed that DFMO has no toxicity on Baby Hamster Kidney-21 (BHK-21) cells and is capable of reducing titers of several different viruses, including Poliovirus, Coxsackievirus B3, Dengue virus and West Nile virus.Based on these results, we started testing whether DFMO could block viral synthesis and reduce viral titers in vivo in zebrafish larvae, while other labs performed few similar tests in mouse and in Drosophila.Initial experiments performed after placing 1-cell stage eggs in DFMO-containing water at different increasing concentrations showed that the compound can be toxic at early stages of development, as the majority of embryos showed developmental defects including a crooked spinal cord, slow blood flow, and edemas.

Figure R4. 1 .

 1 Figure R4.1. Quantification of polyamine depletion and viral replication in control non-treated and DFMO-treated infected larvae. (A) Polyamine quantitation. Inset shows representative chromatogram from each condition. Error bars represent + s.e.m. N = 3 from 1 representative experiment. (B) Viral titers from single larvae homogenates, at 24 and 48 hpi. N = 5 from 1 representative experiment. **P< 0.01.

  identified a prominent role of IFN at early stages post-infection. However, we have shown that IFN alone is not sufficient to prevent the virus from reaching the CNS, as demonstrated by the knock-down of IFN receptors (IFN-R). The characterization of different SINV variants has also revealed how these can modulate the host response and determine very different outcomes for the infected host (section 1 of this Chapter includes a more detailed discussion). At later times post-infection and when the virus has reached the CNS, we have observed an infiltration of neutrophils probably involved in the clearance of the infection (see section 2 of the present Chapter).

  Type I Interferon (IFN) response, with SINV1-GFP inducing IFN at higher levels -Observed mortality rates, with SINV2-mCherry being more lethal -Cell tropism, with SINV1-GFP infecting fewer cell types -Neuroinvasiveness, with SINV2-mCherry reaching the CNS in all larvae, while SINV1-GFP only in about half, when inoculated iv at the same concentration A clear difference between the two variants is their different fluorescent reporter proteins, which have been inserted at different locations in the viral genomes. A previous report by Furuta et al. has shown that replication defective SINV exhibits different properties (in terms of labeling capacity and movement within

2 .EtOH 4 . 7 . 2 .

 2472 ) (61965-026) Fetal Bovine Serum (FBS) (GIBCO) (10270-106) L-Glutamine (L-Glu, 200 mM) (Life Technologies) (25030-024) Purify using phenol-chloroform extraction and ethanol precipitation, and resuspend in 20 µL of ddH 2 O 3. Run 1 µL on a 0.7% agarose gel in 1× TAE In vitro Transcription 1. Mix 1 µg of linearized plasmid (0.5 µg/µL in water) with: a. 10 µL of 2× NTP/Cap, 1 µL TURBO DNase and incubate for 15 mn at 37°C Lithium Chloride Precipitation 1. Add 30 µL ddH 2 O, 25 µL LiCl, 1 µL GlycoBlue TM , mix gently, and keep at least 1 hr at 20°C 2. Centrifuge at 4000 RCF for 15 mn (at least) at 4°C 3. Carefully remove the supernatant and wash the pellet with 200 µL ice-cold 70% Centrifuge at 4000 RCF for 5 mn at 4°C 5. Carefully remove ethanol and dissolve the RNA pellet in 11 µL ddH 2 O 6. Optionally add 1 µL RNaseOUT TM . Store immediately at -80°C or proceed with transfection Run 1 µL on a RNase-free 0.7% agarose gel in 1× TAE BHK Cells Transfection 1. One day prior to transfection, subculture BHK cells in DMEM/5% FBS in a 162-cm 2 flask. Cells must be at ~80% confluence 2. In an eppendorf, mix 500 µL pure opti-MEM* with 60 µL lipofectamine, and incubate for 10 mn (sol.A) 3. In an eppendorf, dilute ~30 µg viral RNA in 500 µL pure opti-MEM, mix with sol.A, and incubate for 20 mn at room temperature (sol. B) 4. Remove the complete DMEM from cells and eventually rinse once with D-PBS 5. Mix sol. B in 10 mL pure opti-MEM*, and add directly to the cells * DMEM can be used too 6. Incubate the cells with the transfection complexes for 2 hrs at 37°C, 5% CO 2 7. Remove the transfection complexes from the flask and add fresh complete DMEM/5% FBS to the cells. 8. Incubate for 18 to 24 hrs, or until complete cytopathic effect (CPE is observed, transfer medium from the flask to a 15-mL conical polypropylene tube Centrifuge at 1000 RCF for 10 mn at 4°C to remove cell debris 3. Transfer virus-containing supernatant into eppendorf tubes, and store at -80°C 4. Titer on BHK cells. In case the titer is too low for the specific need, the virus can be concentrated adding a centrifugation step. In this case, an Amicon® Ultra-15 with a 10K (MW) cutoff should be used. Centrifugation should be performed at 4000 RCF for 15-20 mn at 4°C The following solutions are needed for the plaque assay, in order to determine the virus stock titer: DMEM/5% FBS: 500 mL DMEM, 25 mL FBS, 5 mL L-Glutamine, 5 mL PenStrep DMEM (2×): DMEM (powder), 3.7 g Sodium Bicarbonate, up to 460 mL ddH 2 O, filter (.22µm) and add 20 mL FBS, 10 mL of 100× L-Glutamine, 10 mL of 100× PenStrep. Store for less than 2 months at 4°C

3 . 2 .

 32 Dechorionate the embryos and anesthetize them with tricaine (1x) 4. Remove the head from the rest of the body, using fine forceps and a small cutter Tissue Dissociation and Cell Cultures 1. Transfer the heads in a 1.5 mL eppendorf tube, let them sediment and remove the supernatant Add 500 µL of trypsin, mix gently and incubate for 15 min at 37°C 3. Add 100 µL of FBS to block the trypsin, mix gently and remove the supernatant, after sedimentation 4. Add 1 mL of EPC medium to the eppendorf tube and transfer the content in a 15 mL falcon tube already containing 4 mL of EPC medium mixed with DNAse (100 µL). DNase helps tissue dissociation but it is not essential 5. Grind the tissues with a 5 mL pipette (do not grind more than 10 times); add a 1 mL tip on the P5 pipette and repeat the grinding. At this step, the suspension should appear homogenous 6. Filter the suspension through a 0.40 µm cell strainer 7. Spin down the cells at 90 RCF for 5 min without brakes (at room temperature) 8. Remove the supernatant and wash the cells once in EPC medium (∼4 mL) 9. Spin down the cells at 90 RCF for 5 min without brakes 10. Add 2 mL of EPC medium, resuspend the cells and measure the cell density, using a Bürker chamber. The cell density should be comprised between 5x10 5 and 1x10 6 cells/mL; if a concentration step is needed, proceed again with passage 9. and resuspend the cells in the appropriate volume of EPC medium 11. Plate the cells on coated Ibidi dishes, adding half of the final volume of medium+cells/well (depending on the Ibidi dish being used) and incubate them at 24°C 12. After 1 h (when cells have adhered), add the other half of the medium The medium should be changed every 2-3 days, depending on the state of the culture. It is advisable to change each time only half of the medium, in order to maintain cells viability

Part 1 .

 1 Architecture of Innate Immune Response in Fish: IFNφ, Receptors, General Structure of Pathways Fish IFNs

Fig. 1 .

 1 Fig. 1. Schematic representation of zebrafish IFNs and their receptors. Tridimensional representations of IFNs are from the Protein Data Bank (accession numbers: 3PIV, zebrafish IFNφ1; 3PIW, zebrafish IFNφ2; 3HHC, human IFNλ3; 1AU1, human IFNβ; 1HIG, human IFNγ).

Fig. 2 .

 2 Fig. 2. Schematic representation of IFN signaling pathways in fish. Adaptor molecules are represented in orange, kinases are in green, TRAFs are in purple, transcription factors are in yellow, and IFNs are in red.

  It has been adapted from[START_REF] Chen | Primary neuron culture for nerve growth and axon guidance studies in zebrafish (Danio rerio)[END_REF].

	Plaque Assay The protocol is as follows: Ibidi µ-Slide 8 well	(Biovalley) (80826)
	1. While incubating, prepare DMEM/1% MC mixture (3 mL per well). Mix by 1. Four days prior to the dissociation, set up fish for spawning Ibidi Hi-Q 4 (Biovalley) (MZI00040)
	adding MC to DMEM as MC is very viscous. Plan more, as a part of the mixture 2. The following day, collect the eggs and wash them with regular/Volvic water
	is lost in the pipette 3. Sort the fertilized eggs and discard the non-fertilized ones 25 µg/mL PDL: 25 µL PDL 1 mg/mL, 975 µL D-PBS; PDL is fundamental to
	2. Remove the inoculum from each well, add 3 mL of DMEM/1% MC 4. Wash again with regular/Volvic water and discard excess water. Add 30 µL of increase the adherence of cells to the support
	3. Incubate 2-3 days at 37°C, 5% CO 2 5.25% bleach in 50 mL of regular/Volvic water; gently rotate the falcon; do not 4% D-glucose: 2 g in 50 mL of D-PBS; dissolve carefully and filter the solution
	leave the embryos in bleach for more than 5 minutes with a 0.20 µm cell strainer filter; store at 4°C
	Revealing 5. Wash 2x with regular/Volvic water EPC/10% FBS: 450 mL MEM, 50 mL FBS, 5 mL L-Glu, 5 mL Tryptose, 500
	1. Add 1 mL 4% formaldehyde to cover wells, and incubate at least 30 mn 6. Transfer the embryos in a petri dish with regular/Volvic water with methylene µL PenStrep; if the culture is needed for viral infection studies, the
	2. Remove gently the DMEM/MC/formaldehyde solution, and gently rinse with blue (4 mg/mL -70 µL/L of water) and incubate them at 28.5°C until the day of virus-containing medium must be EPC/2 % FBS
	H 2 O the dissociation
	1.6% MC: 1.6 g of MC, up to 100 mL with H 2 O. Autoclave in 100 mL aliquots, 3. Cover each well with 0.23% CV solution for 5 mn to stain viable cells 2. Add a solution of sterile PDL/PBS (25 µg/mL) in each well of the available
	store at 4°C. A 100 mL bottle is suitable for fifteen 6-well plates 4. Empty out the 0.23% CV solution into a bleach-containing sink, and gently On the day prior to the dissociation: incubation plate, such as for example, a glass-covered Ibidi dish and incubate ∼1
	DMEM/0.8% MC: 6 mL of 1.6% MC in 6 mL of 2× DMEM (per 6-well plate) rinse with H 2 O hr at 37°C
	0.23% CV Solution: 180 mL H 2 O, 45 mL 100% Ethanol, 25 mL 2.3% CV 5. Let dry, count plaques, and calculate titer in plaque forming units per milliliter The following reagents and solutions are needed:
	Solution stock (PFU/mL)
	4% formaldehyde: 650 µL 37% formaldehyde, up to 6 mL with D-PBS (per Ethanol Absolute (EtOH) (VWR) (20820.362)
	6-well plate) 2. Zebrafish Neuronal Cell Cultures Tricaine (25x)	(SIGMA) (E10521)
	2.6% bleach: 270 mL 9.6% sodium hypochlorite, up to 1L with H 2 O D-PBS/Modified (HyClone) (SH30028.02)
	Poly-D-lysine (PDL, 1 mg/mL)	(SIGMA) (P7280-5MG)
	Cells and Virus Dilutions The following protocol was developed at INRA in the group 'Virologie et α-D-glucose (MW: 180.16 g/mol) (Prolabo) (24 370.294)
	1. On the day prior to the plaque assay, split BHK-21 cells into 6-well plate at a Immunologie Moléculaire', under the invaluable guidance of Pierre Boudinot and Difco TM trypsin 250 (100 g) (BD) (215240)
	concentration of 0.7-1 × 10 6 cells per well in 2 mL of 1× DMEM such that cells will be 70-90% confluent on the next day. A 162 cm 2 flask gives ~32 × 10 6 BHK DNase (10000 U) (Roche) (10 776 785 001) Christelle Langevin. It consists in the dissociation of dissected zebrafish embryos, to obtain a MEM (500 mL) (Eurobio) (CXXBHK00-01)
	cells. Incubate overnight at 37°C, 5% CO 2 suspension enriched in neurons and glial cells that is then plated in complete Fetal bovine serum (FBS) (GIBCO) (10270-106)
	2. Thaw the virus to be titered on ice (~30 mn) and prepare serial virus dilutions in medium on a suitable substrate. Preliminary experiments have shown that cells L-glutamine (L-Glu, 200 mM) (PAA) (M11-004)
	1.5 mL eppendorf tubes derived this way can be kept viable for 10-12 days and can be used for various Tryptose (100 mL) (SIGMA) (T8159)
	3. Recover the plate, observe cells, pour medium out of wells, and rinse once with purposes (viral infection studies, gene expression studies, drug screening tests). Penicillin (Pen, 100 U/mL) (Biovalley) (P0142.0100)
	2 mL D-PBS Several zebrafish lines have been used, including: wild-type (WT) AB, Streptomycin (Strep, 100 µg/mL) (Biovalley) (S09470) HuC:eGFP (Tg(elavl3:EGFP) knu3 ) with green neurons, HuC:GAL4 crossed to 4. Infect each well with 250 µL of chosen virus dilution (or DMEM w/o FBS as a negative control) 5. Incubate the plaque for 1 hr at 37°C, agitating every 10 mn UAS:NfsB-mCherry (Tg(elavl3.1:GAL4-VP16) hd5 crossed Pasteur pipette (5 mL, sterile) (VWR) (612-1686) to Tg(UAS-E1b:Eco.NfsB-mCherry) c264 ) with red neurons, Cell strainer filters (0.40 µm) (BD Falcon) (352340) GFAP:eGFP Cell strainer filters (0.20 µm) (Minisart) (16534) (Tg(gfap:EGFP) mi2001 ) with green glial cells. Ibidi µ-Dish 35 mm, high (Biovalley) (81156)

  IRF IFN regulatory factor; dsRNA double-stranded RNA; TIR Toll-interleukin 1 receptor; IRAK interleukin-1Rassociated kinase; VHSV viral hemorrhagic septicemia virus; IHNV infectious hematopoietic necrosis virus; CHIKV Chikungunya virus; NNV nervous necrosis virus; RV rabies virus; VSV vesicular stomatitis virus

Major Histocompatibility Complex: A set of cell surface proteins interacting with either CD4 + T cells or CD8 + T cells to mediate initiation of adaptive immunity.

Viral Latency: The ability of a virus to remain dormant within the host cell, sometimes establishing lifelong occult infection. The viral genome can remain latent either integrated in the host chromosome or as an episome. Stress cellular signals are often promoters of viral genome reactivation, termination of viral latency and re-establishment of infection.

As opposed to Primary neurulation: It involves columnarization of an existing epithelium, and then rolling or folding of the epithelium.

Acknowledgements

We wish to thank Benjamin Obadia for SINV suspensions and plasmids, Michel Brémont for the anti-IHNV antibody, Barbara Rizzi for 3D reconstruction of fli:G/U:RFP larvae, Richard Kuhn for the SINV-capsid antibody, Florence Guivel-Benhassine and Olivier Schwartz for sharing protocols, Aurelie Lunazzi and Maxence Frétaud for technical assistance, Emma Colucci-Guyon and Pedro Hernández for critical reading of the manuscript and Jose Perez for his invaluable care of the fish facility. This work was supported by funding from the European Community's Seventh Framework Programme [FP7-PEOPLE-2011-ITN] under grant agreement no. PITN-GA-2011-289209 for the Marie Curie Initial Training Network FishForPharma, by the Agence Nationale de la Recherche (Zebraflam grant ANR-10-MDI-009), and received institutional support from INRA, the Institut Pasteur, and CNRS.

Acknowledgements

This article is dedicated to the memory of Pierre de Kinkelin, who pioneered the study of fish IFNs and will be dearly missed after passing away in May 2013. The research leading to these results has received funding from the European Community's Seventh Framework Programme [FP7-PEOPLE-2011-ITN] under grant agreement no. PITN-GA-2011-289209 for the Marie-Curie Initial Training Network FishForPharma. N.P. is endowed with a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/60678/2009). This work was also funded by the ANR grant "Zebraflam" (ANR-10-MIDI-009).

LIST OF ABBREVIATIONS

Ab

CHAPTER I. Intravital High-Resolution Microscopy in a Vertebrate Model System Unveils Sindbis Virus Neuroinvasion

Gabriella Passoni 1,2,3 , Christelle Langevin 1 , Nuno Palha 2,3* , Valérie Briolat 2 , Pierre Affaticati 4 , Elodie De Job 4 , Hervé Blanc 5 , Lionel Frangeul 5 , Jean Stéphane Joly 4 , Maria-Carla Saleh 5 , Philippe Herbomel 2 , Pierre Boudinot 1 , Jean-Pierre Levraud 2,3 1. INRA, Virology and Molecular Immunology, Domaine de Vilvert, F-78352, Jouy-en-Josas,  To whom correspondence should be addressed: jean-pierre.levraud@pasteur.fr

Summary

Alphaviruses, such as Chikungunya virus (CHIKV) and Sindbis virus (SINV), are vector-borne pathogens that cause acute illnesses in humans and are sometimes associated with neuropathies, especially in infants and elderly patients. Little is known about their entry mechanism in the central nervous system (CNS). Here we used the optically transparent zebrafish larvae to image in vivo the onset and the progression of the infection caused by SINV. Testing the possible mechanisms of entry in the CNS, we excluded infection of brain endothelium or transport via macrophages, and identified axonal transport from the periphery as the most likely route of access to the CNS. We also found a prominent role of Interferon (IFN) in the control of SINV pathogenesis but not neuroinvasion. These results will facilitate testing therapeutics for the control and prevention of Alphavirus-induced encephalopathy.

Supplemental Information

Supplemental information includes four figures and four movies.

Author Contributions

Conceived and designed the experiments: JPL PB GP CL JSJ MCS PH.

Performed the experiments: GP CL JPL NP VB PA EDJ HB. Analyzed the data: GP JPL PB CL LF. Contributed reagents/materials/analysis tools: PA CS. Wrote the paper: GP JPL PB CL.

Competing Interests Statement

The authors declare no competing interests. The Antiviral Innate Immune Response in Fish: Evolution and Conservation of the IFN System Christelle Langevin 1 , Elina Aleksejeva 1, 2, † , Gabriella Passoni 1, 3, 4, 5, † , Nuno Palha 3, 4, 5 , Jean-Pierre Levraud 3, 4 and Pierre Boudinot