Infection de la barrière hémato-encéphalique par différents virus émergents, neuroinflammation et contexte de circulation

Usutu (USUV) and West-Nile (WNV) virus are emerging arboviruses mainly transmitted by Culex genus mosquitoes. In human, the infection can lead to neurological impairments following invasion of the central nervous system (CNS) by viruses, for example, through the blood-brain barrier (BBB). This thesis project aims to characterize access and infection mechanisms of the BBB by USUV and WNV. Neuroinflammation associated to these infections is an important part of the pathogenesis, consequently immune cell role and their interactions with the BBB have been studied. This work has shown the susceptibility of different cell types of the CNS to different USUV lineages and the susceptibility of murine models. Europe 2 lineage seems to be the more virulent and its effect on the BBB has been compared to the WNV lineage 2 effect, these two lineages being associated to neuronal symptom appearance during human infection. The BBB model used allows the replication of both arbovirus with passage in the CNS, WNV also leads to integrity perturbations of the barrier, and the two viruses are able to induce strong antiviral response with secretion of inflammatory molecules as cytokines (interleukins, interferons), chemokines and cell adhesion molecules. This inflammation induces immune cell recruitment and increases neuroinflammation. Unlike WNV, USUV is able to infect dendritic cells that can be recruited by the BBB and that can be Trojan horses during CNS infection. WNV infected patient sera have been analyzed to highlight the neuroinflammation and to search potential biomarkers of neurological impairments. Infected patients with neurological disorders show high serological concentrations of pro-inflammatory molecules, persisting more than 20 days before infection starting date, and of molecules associated to neurodegenerative or neuroinflammatory diseases. The concentration of TDP-43 could be followed after WNV infection in order to anticipate the consequences of its aggregation that can promote neurodegenerative disease development, for example. In a “One Health” approach, this work also studies circulation of these viruses in the south of France. Seroprevalence assays have been done to determine USUV and WNV exposition in vectors, reservoirs and other hosts. Finally, to investigate emerging virus that cause neuronal disorders, a study of the BBB infection by SARS-CoV-2 has been accomplished. Indeed, this virus can cause neuronal symptoms with possible sequels. Results suggest a poor direct infection of the barrier. Taken together, this work allow a better understanding of BBB viral infections and neuroinflammation, in a context of emergence associated to public health concerns that highlights importance of these studies.Les virus Usutu (USUV) et West-Nile (WNV) sont des arbovirus émergents transmis principalement par des moustiques du genre Culex. Chez l’humain l’infection peut entrainer des atteintes neurologiques suite à un accès des virus au système nerveux central (SNC) notamment par le passage de la barrière hémato-encéphalique (BHE). Ce projet de thèse s’intéresse à la caractérisation des mécanismes de passage et d’infection de la BHE par USUV et WNV. La neuroinflammation associée à ces infections étant une part importante de la pathogénèse, le rôle des cellules immunes et leur interaction avec la BHE ont aussi été étudiés. Ce travail a permis de mettre en évidence la susceptibilité de plusieurs types cellulaires neuronaux aux différentes lignées d’USUV ainsi que celle de modèles murins. La lignée Europe 2 semble être la plus virulente et son effet sur la BHE a été comparé avec celui de la lignée 2 de WNV, deux lignées associées à l’apparition de symptômes neuronaux lors d’infection humaine. Le modèle de BHE étudié permet la réplication des deux arbovirus avec un passage dans le SNC, WNV conduit aussi à une perturbation de l’intégrité de la barrière et les deux virus sont capable d’induire une forte réponse antivirale avec la sécrétion de molécules inflammatoires comme des cytokines (interleukines, interférons), des chimiokines et des molécules d’adhésion cellulaires. Cette inflammation induit le recrutement de cellules immunes amplifiant la neuroinflammation. D’autre part, USUV est capable d’infecter des cellules dendritiques qui seront aussi recrutées par la BHE à la différence de WNV, et peuvent être des chevaux de Troie lors de l’infection du SNC. Des sérums de patients ayant été infectés par le virus WNV ont été analysés afin de mettre en évidence la neuroinflammation et de trouver de potentiel biomarqueurs d’atteintes neuronales. Les patients infectés présentant des atteintes neurologiques montrent ainsi de fortes concentrations sériques de molécules pro-inflammatoires, persistantes plus de 20 jours après le début de l’infection, et la présence de molécules associées à des maladies neurodégénératives ou neuroinflammatoire. Par exemple, la concentration de TDP-43 pourrait être suivie après une infection par WNV afin d’anticiper les conséquences de son agrégation pouvant favoriser le développement de maladies neurodégénératives. Dans une démarche de « santé globale », ces travaux se sont aussi intéressés à la circulation de ces virus dans le sud de la France. Des tests de séroprévalence ont été menés afin de déterminer l’exposition des vecteurs, des réservoirs et d’autres hôtes à USUV et WNV. Enfin, en s’intéressant aux virus émergents responsables de troubles neuronaux, un travail de recherche sur l’infection de la BHE par le virus SARS-CoV-2 a été réalisé. En effet, ce virus est capable d’induire des symptômes neuronaux pouvant entrainer des séquelles. Les résultats suggèrent une faible infection directe de la barrière. L’ensemble de ces travaux permettent de mieux appréhender les infections virales de la BHE et la neuroinflammation, cela dans un contexte d’émergence avec des problématiques de santé publique soulevant l’importance de ces études

Blood-brain barrier infection by different emerging viruses, neuroinflammation, and circulation context

Les virus Usutu (USUV) et West-Nile (WNV) sont des arbovirus émergents transmis principalement par des moustiques du genre Culex. Chez l’humain l’infection peut entrainer des atteintes neurologiques suite à un accès des virus au système nerveux central (SNC) notamment par le passage de la barrière hémato-encéphalique (BHE). Ce projet de thèse s’intéresse à la caractérisation des mécanismes de passage et d’infection de la BHE par USUV et WNV. La neuroinflammation associée à ces infections étant une part importante de la pathogénèse, le rôle des cellules immunes et leur interaction avec la BHE ont aussi été étudiés. Ce travail a permis de mettre en évidence la susceptibilité de plusieurs types cellulaires neuronaux aux différentes lignées d’USUV ainsi que celle de modèles murins. La lignée Europe 2 semble être la plus virulente et son effet sur la BHE a été comparé avec celui de la lignée 2 de WNV, deux lignées associées à l’apparition de symptômes neuronaux lors d’infection humaine. Le modèle de BHE étudié permet la réplication des deux arbovirus avec un passage dans le SNC, WNV conduit aussi à une perturbation de l’intégrité de la barrière et les deux virus sont capable d’induire une forte réponse antivirale avec la sécrétion de molécules inflammatoires comme des cytokines (interleukines, interférons), des chimiokines et des molécules d’adhésion cellulaires. Cette inflammation induit le recrutement de cellules immunes amplifiant la neuroinflammation. D’autre part, USUV est capable d’infecter des cellules dendritiques qui seront aussi recrutées par la BHE à la différence de WNV, et peuvent être des chevaux de Troie lors de l’infection du SNC. Des sérums de patients ayant été infectés par le virus WNV ont été analysés afin de mettre en évidence la neuroinflammation et de trouver de potentiel biomarqueurs d’atteintes neuronales. Les patients infectés présentant des atteintes neurologiques montrent ainsi de fortes concentrations sériques de molécules pro-inflammatoires, persistantes plus de 20 jours après le début de l’infection, et la présence de molécules associées à des maladies neurodégénératives ou neuroinflammatoire. Par exemple, la concentration de TDP-43 pourrait être suivie après une infection par WNV afin d’anticiper les conséquences de son agrégation pouvant favoriser le développement de maladies neurodégénératives. Dans une démarche de « santé globale », ces travaux se sont aussi intéressés à la circulation de ces virus dans le sud de la France. Des tests de séroprévalence ont été menés afin de déterminer l’exposition des vecteurs, des réservoirs et d’autres hôtes à USUV et WNV. Enfin, en s’intéressant aux virus émergents responsables de troubles neuronaux, un travail de recherche sur l’infection de la BHE par le virus SARS-CoV-2 a été réalisé. En effet, ce virus est capable d’induire des symptômes neuronaux pouvant entrainer des séquelles. Les résultats suggèrent une faible infection directe de la barrière. L’ensemble de ces travaux permettent de mieux appréhender les infections virales de la BHE et la neuroinflammation, cela dans un contexte d’émergence avec des problématiques de santé publique soulevant l’importance de ces études.Usutu (USUV) and West-Nile (WNV) virus are emerging arboviruses mainly transmitted by Culex genus mosquitoes. In human, the infection can lead to neurological impairments following invasion of the central nervous system (CNS) by viruses, for example, through the blood-brain barrier (BBB). This thesis project aims to characterize access and infection mechanisms of the BBB by USUV and WNV. Neuroinflammation associated to these infections is an important part of the pathogenesis, consequently immune cell role and their interactions with the BBB have been studied. This work has shown the susceptibility of different cell types of the CNS to different USUV lineages and the susceptibility of murine models. Europe 2 lineage seems to be the more virulent and its effect on the BBB has been compared to the WNV lineage 2 effect, these two lineages being associated to neuronal symptom appearance during human infection. The BBB model used allows the replication of both arbovirus with passage in the CNS, WNV also leads to integrity perturbations of the barrier, and the two viruses are able to induce strong antiviral response with secretion of inflammatory molecules as cytokines (interleukins, interferons), chemokines and cell adhesion molecules. This inflammation induces immune cell recruitment and increases neuroinflammation. Unlike WNV, USUV is able to infect dendritic cells that can be recruited by the BBB and that can be Trojan horses during CNS infection. WNV infected patient sera have been analyzed to highlight the neuroinflammation and to search potential biomarkers of neurological impairments. Infected patients with neurological disorders show high serological concentrations of pro-inflammatory molecules, persisting more than 20 days before infection starting date, and of molecules associated to neurodegenerative or neuroinflammatory diseases. The concentration of TDP-43 could be followed after WNV infection in order to anticipate the consequences of its aggregation that can promote neurodegenerative disease development, for example. In a “One Health” approach, this work also studies circulation of these viruses in the south of France. Seroprevalence assays have been done to determine USUV and WNV exposition in vectors, reservoirs and other hosts. Finally, to investigate emerging virus that cause neuronal disorders, a study of the BBB infection by SARS-CoV-2 has been accomplished. Indeed, this virus can cause neuronal symptoms with possible sequels. Results suggest a poor direct infection of the barrier. Taken together, this work allow a better understanding of BBB viral infections and neuroinflammation, in a context of emergence associated to public health concerns that highlights importance of these studies

Infection de la barrière hémato-encéphalique par différents virus émergents, neuroinflammation et contexte de circulation

Usutu (USUV) and West-Nile (WNV) virus are emerging arboviruses mainly transmitted by Culex genus mosquitoes. In human, the infection can lead to neurological impairments following invasion of the central nervous system (CNS) by viruses, for example, through the blood-brain barrier (BBB). This thesis project aims to characterize access and infection mechanisms of the BBB by USUV and WNV. Neuroinflammation associated to these infections is an important part of the pathogenesis, consequently immune cell role and their interactions with the BBB have been studied. This work has shown the susceptibility of different cell types of the CNS to different USUV lineages and the susceptibility of murine models. Europe 2 lineage seems to be the more virulent and its effect on the BBB has been compared to the WNV lineage 2 effect, these two lineages being associated to neuronal symptom appearance during human infection. The BBB model used allows the replication of both arbovirus with passage in the CNS, WNV also leads to integrity perturbations of the barrier, and the two viruses are able to induce strong antiviral response with secretion of inflammatory molecules as cytokines (interleukins, interferons), chemokines and cell adhesion molecules. This inflammation induces immune cell recruitment and increases neuroinflammation. Unlike WNV, USUV is able to infect dendritic cells that can be recruited by the BBB and that can be Trojan horses during CNS infection. WNV infected patient sera have been analyzed to highlight the neuroinflammation and to search potential biomarkers of neurological impairments. Infected patients with neurological disorders show high serological concentrations of pro-inflammatory molecules, persisting more than 20 days before infection starting date, and of molecules associated to neurodegenerative or neuroinflammatory diseases. The concentration of TDP-43 could be followed after WNV infection in order to anticipate the consequences of its aggregation that can promote neurodegenerative disease development, for example. In a “One Health” approach, this work also studies circulation of these viruses in the south of France. Seroprevalence assays have been done to determine USUV and WNV exposition in vectors, reservoirs and other hosts. Finally, to investigate emerging virus that cause neuronal disorders, a study of the BBB infection by SARS-CoV-2 has been accomplished. Indeed, this virus can cause neuronal symptoms with possible sequels. Results suggest a poor direct infection of the barrier. Taken together, this work allow a better understanding of BBB viral infections and neuroinflammation, in a context of emergence associated to public health concerns that highlights importance of these studies.Les virus Usutu (USUV) et West-Nile (WNV) sont des arbovirus émergents transmis principalement par des moustiques du genre Culex. Chez l’humain l’infection peut entrainer des atteintes neurologiques suite à un accès des virus au système nerveux central (SNC) notamment par le passage de la barrière hémato-encéphalique (BHE). Ce projet de thèse s’intéresse à la caractérisation des mécanismes de passage et d’infection de la BHE par USUV et WNV. La neuroinflammation associée à ces infections étant une part importante de la pathogénèse, le rôle des cellules immunes et leur interaction avec la BHE ont aussi été étudiés. Ce travail a permis de mettre en évidence la susceptibilité de plusieurs types cellulaires neuronaux aux différentes lignées d’USUV ainsi que celle de modèles murins. La lignée Europe 2 semble être la plus virulente et son effet sur la BHE a été comparé avec celui de la lignée 2 de WNV, deux lignées associées à l’apparition de symptômes neuronaux lors d’infection humaine. Le modèle de BHE étudié permet la réplication des deux arbovirus avec un passage dans le SNC, WNV conduit aussi à une perturbation de l’intégrité de la barrière et les deux virus sont capable d’induire une forte réponse antivirale avec la sécrétion de molécules inflammatoires comme des cytokines (interleukines, interférons), des chimiokines et des molécules d’adhésion cellulaires. Cette inflammation induit le recrutement de cellules immunes amplifiant la neuroinflammation. D’autre part, USUV est capable d’infecter des cellules dendritiques qui seront aussi recrutées par la BHE à la différence de WNV, et peuvent être des chevaux de Troie lors de l’infection du SNC. Des sérums de patients ayant été infectés par le virus WNV ont été analysés afin de mettre en évidence la neuroinflammation et de trouver de potentiel biomarqueurs d’atteintes neuronales. Les patients infectés présentant des atteintes neurologiques montrent ainsi de fortes concentrations sériques de molécules pro-inflammatoires, persistantes plus de 20 jours après le début de l’infection, et la présence de molécules associées à des maladies neurodégénératives ou neuroinflammatoire. Par exemple, la concentration de TDP-43 pourrait être suivie après une infection par WNV afin d’anticiper les conséquences de son agrégation pouvant favoriser le développement de maladies neurodégénératives. Dans une démarche de « santé globale », ces travaux se sont aussi intéressés à la circulation de ces virus dans le sud de la France. Des tests de séroprévalence ont été menés afin de déterminer l’exposition des vecteurs, des réservoirs et d’autres hôtes à USUV et WNV. Enfin, en s’intéressant aux virus émergents responsables de troubles neuronaux, un travail de recherche sur l’infection de la BHE par le virus SARS-CoV-2 a été réalisé. En effet, ce virus est capable d’induire des symptômes neuronaux pouvant entrainer des séquelles. Les résultats suggèrent une faible infection directe de la barrière. L’ensemble de ces travaux permettent de mieux appréhender les infections virales de la BHE et la neuroinflammation, cela dans un contexte d’émergence avec des problématiques de santé publique soulevant l’importance de ces études

West Nile Virus Neuroinfection in Humans: Peripheral Biomarkers of Neuroinflammation and Neuronal Damage

International audienceAmong emerging arthropod-borne viruses (arbovirus), West Nile virus (WNV) is a flavivirus that can be associated with severe neuroinvasive infections in humans. In 2018, the European WNV epidemic resulted in over 2000 cases, representing the most important arboviral epidemic in the European continent. Characterization of inflammation and neuronal biomarkers released during WNV infection, especially in the context of neuronal impairments, could provide insight into the development of predictive tools that could be beneficial for patient outcomes. We first analyzed the inflammatory signature in the serum of WNV-infected mice and found increased concentrations of several inflammatory cytokines. We next analyzed serum and cerebrospinal-fluid (CSF) samples from a cohort of patients infected by WNV between 2018 and 2019 in Hungary to quantify a large panel of inflammatory cytokines and neurological factors. We found higher levels of inflammatory cytokines (e.g., IL4, IL6, and IL10) and neuronal factors (e.g., BDNF, GFAP, MIF, TDP-43) in the sera of WNV-infected patients with neuroinvasive disease. Furthermore, the serum inflammatory profile of these patients persisted for several weeks after initial infection, potentially leading to long-term sequelae and having a deleterious effect on brain neurovasculature. This work suggests that early signs of increased serum concentrations of inflammatory cytokines and neuronal factors could be a signature underlying the development of severe neurological impairments. Biomarkers could play an important role in patient monitoring to improve care and prevent undesirable outcomes

Role of Dendritic Cells in Viral Brain Infections

International audienceTo gain access to the brain, a so-called immune-privileged organ due to its physical separation from the blood stream, pathogens and particularly viruses have been selected throughout evolution for their use of specific mechanisms. They can enter the central nervous system through direct infection of nerves or cerebral barriers or through cell-mediated transport. Indeed, peripheral lymphoid and myeloid immune cells can interact with the blood–brain and the blood–cerebrospinal fluid barriers and allow viral brain access using the “Trojan horse” mechanism. Among immune cells, at the frontier between innate and adaptive immune responses, dendritic cells (DCs) can be pathogen carriers, regulate or exacerbate antiviral responses and neuroinflammation, and therefore be involved in viral transmission and spread. In this review, we highlight an important contribution of DCs in the development and the consequences of viral brain infections

Differential effects of Usutu and West Nile viruses on neuroinflammation, immune cell recruitment and blood–brain barrier integrity

ABSTRACTUsutu (USUV) and West Nile (WNV) viruses are two closely related Flavivirus belonging to Japanese encephalitis virus serogroup. Evidence of increased circulation of these two arboviruses now exist in Europe. Neurological disorders are reported in humans mainly for WNV, despite the fact that the interaction and effects of viral infections on the neurovasculature are poorly described, notably for USUV. Using a human in vitro blood–brain barrier (BBB) and a mouse model, this study characterizes and compares the cerebral endothelial cell permissiveness, innate immunity and inflammatory responses and immune cell recruitment during infection by USUV and WNV. Both viruses are able to infect and cross the human BBB but with different consequences. We observed that WNV infects BBB cells resulting in significant endothelium impairment, potent neuroinflammation and immune cell recruitment, in agreement with previous studies. USUV, despite being able to infect BBB cells with higher replication rate than WNV, does not strongly affect endothelium integrity. Importantly, USUV also induces neuroinflammation, immune cell recruitment such as T lymphocytes, monocytes and dendritic cells (DCs) and was able to infect dendritic cells (DCs) more efficiently compared to WNV, with greater propensity for BBB recruitment. DCs may have differential roles for neuroinvasion of the two related viruses

Human Usutu Virus Infection with Atypical Neurologic Presentation, Montpellier, France, 2016

Infection with Usutu virus (USUV) has been recently associated with neurologic disorders, such as encephalitis or meningoencephalitis, in humans. These findings indicate that USUV is a potential health threat. We report an acute human infection with USUV in France putatively associated with a clinical diagnosis of idiopathic facial paralysis

Evidence of Exposure to USUV and WNV in Zoo Animals in France

International audienceWest Nile virus (WNV) and Usutu virus (USUV) are zoonotic arboviruses. These flaviviruses are mainly maintained in the environment through an enzootic cycle involving mosquitoes and birds. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Nevertheless, there is little data regarding the involvement of other mammals in the epidemiology of these arboviruses. In this study, we performed a serosurvey to assess exposure to these viruses in captive birds and mammals in a zoo situated in the south of France, an area described for the circulation of these two viruses. A total of 411 samples comprising of 70 species were collected over 16 years from 2003 to 2019. The samples were first tested by a competitive enzyme-linked immunosorbent assay. The positive sera were then tested using virus-specific microneutralization tests against USUV and WNV. USUV seroprevalence in birds was 10 times higher than that of WNV (14.59% versus 1.46%, respectively). Among birds, greater rhea (Rhea Americana) and common peafowl (Pavo cristatus) exhibited the highest USUV seroprevalence. Infections occurred mainly between 2016–2018 corresponding to a period of high circulation of these viruses in Europe. In mammalian species, antibodies against WNV were detected in one dama gazelle (Nanger dama) whereas serological evidence of USUV infection was observed in several Canidae, especially in African wild dogs (Lycaon pictus). Our study helps to better understand the exposure of captive species to WNV and USUV and to identify potential host species to include in surveillance programs in zoos

SARS-CoV-2 Poorly Replicates in Cells of the Human Blood-Brain Barrier Without Associated Deleterious Effects

International audienceVarious neurological symptoms have been associated to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection including headache, fever, anosmia, ageusia, but also, encephalitis, Guillain-Barre syndrome and ischemic stroke. Responsible for the current coronavirus disease (COVID-19) pandemic, SARS-CoV-2 may access and affect the central nervous system (CNS) by several pathways such as axonal retrograde transport or through interaction with the blood-brain barrier (BBB) or blood-cerebrospinal fluid (CSF) barrier. Here, we explored the molecular and cellular effects of direct SARS-CoV-2 infection of human BBB cells. We observed low replication of SARS-CoV-2 that was accompanied by very moderate inflammatory response. Using a human in vitro BBB model, we also described low replication levels without strong inflammatory response or modulation of endothelium integrity. Finally, using serum samples from COVID-19 patients, we highlighted strong concentrations of pro-inflammatory factors that did not perturb BBB integrity after short term exposure. Altogether, our results show that the main mechanism of brain access following SARS-CoV-2 infection does not seem to be directed by brain infection through endothelial cells

Deleterious effect of Usutu virus on human neural cells

In the last decade, the number of emerging Flaviviruses described worldwide has increased considerably. Among them Zika virus (ZIKV) and Usutu virus (USUV) are African mosquitoborne viruses that recently emerged. Recently, ZIKV has been intensely studied due to major outbreaks associated with neonatal death and birth defects, as well as neurological symptoms. USUV pathogenesis remains largely unexplored, despite significant human and veterinary associated disorders. Circulation of USUV in Africa was documented more than 50 years ago, and it emerged in Europe two decades ago, causing massive bird mortality. More recently, USUV has been described to be associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting USUV as a potential health threat. The aim of this study was to evaluate the ability of USUV to infect neuronal cells. Our results indicate that USUV efficiently infects neurons, astrocytes, microglia and IPSc-derived human neuronal stem cells. When compared to ZIKV, USUV led to a higher infection rate, viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence