Viral Entry Properties Required for Fitness in Humans Are Lost through Rapid Genomic Change during Viral Isolation

Human parainfluenza viruses cause a large burden of human respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus 3 (HPIV-3) evolves in culture. Cultured viruses differ in their properties compared to clinical strains. We present a genome-wide survey of HPIV-3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). Nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins—the receptor binding protein hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV-3 primary culture isolates and 23 HPIV-3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN’s dimerization/F-interaction site, a site critical for activation of viral fusion. Alterations in HN dimer interface residues known to favor infection in culture occurred within 4 days (H552 and N556). A novel cluster of residues at a different face of the HN dimer interface emerged (P241 and R242) and imply a role in HPIV-3-mediated fusion. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV-3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity. These results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the HN-F complex required for fitness by circulating viruses. IMPORTANCE Human parainfluenza virus 3 is an important cause of morbidity and mortality among infants, the immunocompromised, and the elderly. Using deep genomic sequencing of HPIV-3-positive clinical material and its subsequent viral isolate, we discover a number of known and novel coding mutations in the main HPIV-3 attachment protein HN during brief exposure to immortalized cells. These mutations significantly alter function of the fusion complex, increasing fusion promotion by HN as well as generally decreasing neuraminidase activity and increasing HN-receptor engagement. These results show that viruses may evolve rapidly in culture even during primary isolation of the virus and before the first passage and reveal features of fitness for humans that are obscured by rapid adaptation to laboratory conditions

Physiopathology of central nervous infection with respiratory viruses

Les virus respiratoires aéroportés sont particulièrement préoccupants du fait de la difficulté de contrôler leur transmission. Parmi ces virus, le virus du syndrome respiratoire aigu sévère 2 (SARS-CoV-2), le virus de la rougeole (VR) et les Henipavirus Nipah (NiV) et Hendra (HeV) peuvent infecter également le système nerveux central (SNC) chez l’homme et provoquent alors souvent des encéphalites létales. Par exemple, le SARS-CoV-2, responsable de la pandémie de COVID-19, entraine un syndrome respiratoire aigu sévère et des atteintes neurologiques. De son coté, et malgré un vaccin efficace, la rougeole connait une réémergence inquiétante et cause la mort de plus de 200 000 personnes par an. Le VR peut entrainer des encéphalites rougeoleuses à corps d’inclusion (MIBE) dans un contexte d’immunodéficience ou une panencéphalite sclérosante subaiguë (PESS) parfois des décennies après l’exposition au virus chez des patients immunocompétents. NiV et HeV sont des Paramyxovirus zoonotiques hautement pathogènes du genre Henipavirus. Malgré le faible nombre de cas humains recensés depuis leur émergence à la fin des années 1990, les NiV et HeV sont classés parmi les huit pathogènes prioritaires pour la recherche par l’Organisation Mondiale de la Santé en raison de leur fort potentiel pandémique. Certaines souches sont mortelles dans plus de 70% des cas en moyenne. À ce jour il n’existe pas de traitement efficace commercialisé pour traiter ces infections virales chez l’homme. De plus, les étapes précoces de l’infection du SNC par ces virus restent peu documentées car la majorité des données proviennent d’analyses réalisées post mortem. L’objectif global de cette thèse a été d’identifier des facteurs influençant l’invasion du SNC par ces virus. Le tropisme initial, la dissémination, ainsi que l’implication des glycoprotéines virales de surface et l’évolution génétique virale ont été analysées pour le SARS-CoV-2, le VR et plusieurs souches d’Henipavirus à l’échelle organique, cellulaire et moléculaire. Deux nouveaux modèles de cultures organotypiques de poumons et de tronc cérébral chez le hamster ont été développés et caractérisés. Ces modèles ex vivo sont susceptibles à l’infection par le SARS-CoV-2 et par le NiV. En revanche, un mutant hyperfusogène du VR, pourtant capable de fusionner en l’absence de récepteur connu, n’infecte que les cultures de cerveau. Ces cultures organotypiques ont permis de valider le tropisme initial du SARS-CoV-2 dans les poumons et démontré la permissivité de certains neurones dans le cerveau. Ces modèles ont également permis d’établir que l’infection par le SARS-CoV-2 induit une réponse interféron spécifique et une réponse immunitaire innée, ainsi qu’une mort cellulaire par apoptose, nécroptose et pyroptose dans ces organes. Enfin, ces cultures organotypiques ont montré leur pertinence dans la validation de l’effet d’antiviraux. L’étude de VR portant des mutations dans leur protéine de fusion observées lors d’encéphalites rougeoleuses a montré l’importance du caractère hyperfusogène de ces mutants pour se disséminer dans le SNC pourtant dépourvu de récepteurs connus. Des différences dans la machinerie de fusion de trois souches pathogènes d’Henipavirus ont aussi été identifiées et analysées.Grace aux cultures organotypiques cérébrales de hamster et de souris transgéniques plusieurs candidats antiviraux ont été testés pour bloquer la dissémination du VR sauvage et de variants neuroinvasifs, mais aussi du NiV et du SARS-CoV-2. Ces résultats donnent des perspectives nouvelles d’utilisation de ces modèles ex vivo pour étudier l’infection par des virus émergents et pour évaluer l’efficacité de traitements en amont de validation in vivo. L’étude comparative de l’infection des cultures organotypiques par ces virus respiratoires à pathogénicité variable a illustré comment la machinerie de fusion peut influencer la dissémination virale dans le cerveau.Airborne respiratory viruses are of particular concern because of the difficulty in controlling their transmission. Among these viruses, severe acute respiratory syndrome virus 2 (SARS-CoV-2), measles virus (MeV) and Henipavirus Nipah (NiV) and Hendra (HeV) can also infect the central nervous system (CNS) in humans and cause lethal encephalitis. For example, SARS-CoV-2, responsible for the COVID-19 pandemic, causes severe acute respiratory syndrome and neurological syndromes. Despite an effective vaccine, measles is reemerging and still responsible of more than 200 000 deaths per year. MeV can lead to measles inclusion-body encephalitis (MIBE) in immunocompromised patients or sub-acute sclerosing panencephalitis (SSPE) sometimes decades after exposure to the virus in immunocompetent patients. NiV and HeV are highly pathogenic zoonotic Paramyxoviruses that belong to the genus Henipavirus. Despite the low number of human cases recorded since their emergence in the late 1990s, NiV and HeV are classified among the top eight pathogens to prioritize for research and development in public health emergency contexts by the World Health Organization because of their high pandemic potential. Some strains are fatal in more than 70% of cases. To date, there is no effective commercialized treatment to cure these viral infections in human. Moreover, the early stages of the CNS infection by these three viruses remain poorly documented because most of the data come from post-mortem analyzes. The overall objective of this thesis was to identify factors influencing the CNS invasion by these viruses. The initial tropism, the dissemination, as well as the involvement of viral surface glycoproteins and viral genetic evolution were analyzed for SARS-CoV-2, MeV and several Henipavirus strains at the organic, cellular, and molecular levels. Two new models of organotypic cultures from hamster brainstem and lung have been developed and characterized. These ex vivo models are susceptible to the infection with SARS-CoV-2 and NiV. In contrast, a hyperfusogenic MeV mutant able to fuse in absence of known receptor, could only infect brain cultures. In these organotypic cultures the initial tropism of SARS-CoV-2 in the lungs was validated and the permissiveness of certain neurons in the brain was demonstrated. The results also showed that SARS-CoV-2 infection induces specific interferon and innate immune responses, along with cell death by apoptosis, necroptosis, and pyroptosis. Finally, these organotypic cultures have shown their relevance in validating the effect of antiviral treatments. The study of MeV carrying mutations in their fusion protein observed in measles encephalitis cases has shown the importance of the hyperfusogenic property to disseminate within the CNS, which lacks the expression of known receptors. Differences in the fusion machinery of three pathogenic Henipavirus strains were also identified and analyzed.Several antiviral candidates have been tested in organotypic brain cultures from hamsters and transgenic mice to block the dissemination of wild-type MeV and neuroinvasive MeV variants, but also of NiV and SARS-CoV-2. These results pave the way for the use of these ex vivo models to study newly emerged viruses’ pathogenesis and assess the efficacy of candidate antivirals before in vivo validation. The comparative study of the organotypic culture infections by these respiratory viruses with high pathogenicity differences illustrated how the fusion machinery can influence the viral dissemination in the brain

Physiopathologie de l'infection du système nerveux central par des virus respiratoires

Airborne respiratory viruses are of particular concern because of the difficulty in controlling their transmission. Among these viruses, severe acute respiratory syndrome virus 2 (SARS-CoV-2), measles virus (MeV) and Henipavirus Nipah (NiV) and Hendra (HeV) can also infect the central nervous system (CNS) in humans and cause lethal encephalitis. For example, SARS-CoV-2, responsible for the COVID-19 pandemic, causes severe acute respiratory syndrome and neurological syndromes. Despite an effective vaccine, measles is reemerging and still responsible of more than 200 000 deaths per year. MeV can lead to measles inclusion-body encephalitis (MIBE) in immunocompromised patients or sub-acute sclerosing panencephalitis (SSPE) sometimes decades after exposure to the virus in immunocompetent patients. NiV and HeV are highly pathogenic zoonotic Paramyxoviruses that belong to the genus Henipavirus. Despite the low number of human cases recorded since their emergence in the late 1990s, NiV and HeV are classified among the top eight pathogens to prioritize for research and development in public health emergency contexts by the World Health Organization because of their high pandemic potential. Some strains are fatal in more than 70% of cases. To date, there is no effective commercialized treatment to cure these viral infections in human. Moreover, the early stages of the CNS infection by these three viruses remain poorly documented because most of the data come from post-mortem analyzes. The overall objective of this thesis was to identify factors influencing the CNS invasion by these viruses. The initial tropism, the dissemination, as well as the involvement of viral surface glycoproteins and viral genetic evolution were analyzed for SARS-CoV-2, MeV and several Henipavirus strains at the organic, cellular, and molecular levels. Two new models of organotypic cultures from hamster brainstem and lung have been developed and characterized. These ex vivo models are susceptible to the infection with SARS-CoV-2 and NiV. In contrast, a hyperfusogenic MeV mutant able to fuse in absence of known receptor, could only infect brain cultures. In these organotypic cultures the initial tropism of SARS-CoV-2 in the lungs was validated and the permissiveness of certain neurons in the brain was demonstrated. The results also showed that SARS-CoV-2 infection induces specific interferon and innate immune responses, along with cell death by apoptosis, necroptosis, and pyroptosis. Finally, these organotypic cultures have shown their relevance in validating the effect of antiviral treatments. The study of MeV carrying mutations in their fusion protein observed in measles encephalitis cases has shown the importance of the hyperfusogenic property to disseminate within the CNS, which lacks the expression of known receptors. Differences in the fusion machinery of three pathogenic Henipavirus strains were also identified and analyzed.Several antiviral candidates have been tested in organotypic brain cultures from hamsters and transgenic mice to block the dissemination of wild-type MeV and neuroinvasive MeV variants, but also of NiV and SARS-CoV-2. These results pave the way for the use of these ex vivo models to study newly emerged viruses’ pathogenesis and assess the efficacy of candidate antivirals before in vivo validation. The comparative study of the organotypic culture infections by these respiratory viruses with high pathogenicity differences illustrated how the fusion machinery can influence the viral dissemination in the brain.Les virus respiratoires aéroportés sont particulièrement préoccupants du fait de la difficulté de contrôler leur transmission. Parmi ces virus, le virus du syndrome respiratoire aigu sévère 2 (SARS-CoV-2), le virus de la rougeole (VR) et les Henipavirus Nipah (NiV) et Hendra (HeV) peuvent infecter également le système nerveux central (SNC) chez l’homme et provoquent alors souvent des encéphalites létales. Par exemple, le SARS-CoV-2, responsable de la pandémie de COVID-19, entraine un syndrome respiratoire aigu sévère et des atteintes neurologiques. De son coté, et malgré un vaccin efficace, la rougeole connait une réémergence inquiétante et cause la mort de plus de 200 000 personnes par an. Le VR peut entrainer des encéphalites rougeoleuses à corps d’inclusion (MIBE) dans un contexte d’immunodéficience ou une panencéphalite sclérosante subaiguë (PESS) parfois des décennies après l’exposition au virus chez des patients immunocompétents. NiV et HeV sont des Paramyxovirus zoonotiques hautement pathogènes du genre Henipavirus. Malgré le faible nombre de cas humains recensés depuis leur émergence à la fin des années 1990, les NiV et HeV sont classés parmi les huit pathogènes prioritaires pour la recherche par l’Organisation Mondiale de la Santé en raison de leur fort potentiel pandémique. Certaines souches sont mortelles dans plus de 70% des cas en moyenne. À ce jour il n’existe pas de traitement efficace commercialisé pour traiter ces infections virales chez l’homme. De plus, les étapes précoces de l’infection du SNC par ces virus restent peu documentées car la majorité des données proviennent d’analyses réalisées post mortem. L’objectif global de cette thèse a été d’identifier des facteurs influençant l’invasion du SNC par ces virus. Le tropisme initial, la dissémination, ainsi que l’implication des glycoprotéines virales de surface et l’évolution génétique virale ont été analysées pour le SARS-CoV-2, le VR et plusieurs souches d’Henipavirus à l’échelle organique, cellulaire et moléculaire. Deux nouveaux modèles de cultures organotypiques de poumons et de tronc cérébral chez le hamster ont été développés et caractérisés. Ces modèles ex vivo sont susceptibles à l’infection par le SARS-CoV-2 et par le NiV. En revanche, un mutant hyperfusogène du VR, pourtant capable de fusionner en l’absence de récepteur connu, n’infecte que les cultures de cerveau. Ces cultures organotypiques ont permis de valider le tropisme initial du SARS-CoV-2 dans les poumons et démontré la permissivité de certains neurones dans le cerveau. Ces modèles ont également permis d’établir que l’infection par le SARS-CoV-2 induit une réponse interféron spécifique et une réponse immunitaire innée, ainsi qu’une mort cellulaire par apoptose, nécroptose et pyroptose dans ces organes. Enfin, ces cultures organotypiques ont montré leur pertinence dans la validation de l’effet d’antiviraux. L’étude de VR portant des mutations dans leur protéine de fusion observées lors d’encéphalites rougeoleuses a montré l’importance du caractère hyperfusogène de ces mutants pour se disséminer dans le SNC pourtant dépourvu de récepteurs connus. Des différences dans la machinerie de fusion de trois souches pathogènes d’Henipavirus ont aussi été identifiées et analysées.Grace aux cultures organotypiques cérébrales de hamster et de souris transgéniques plusieurs candidats antiviraux ont été testés pour bloquer la dissémination du VR sauvage et de variants neuroinvasifs, mais aussi du NiV et du SARS-CoV-2. Ces résultats donnent des perspectives nouvelles d’utilisation de ces modèles ex vivo pour étudier l’infection par des virus émergents et pour évaluer l’efficacité de traitements en amont de validation in vivo. L’étude comparative de l’infection des cultures organotypiques par ces virus respiratoires à pathogénicité variable a illustré comment la machinerie de fusion peut influencer la dissémination virale dans le cerveau

Physiopathologie de l'infection du système nerveux central par des virus respiratoires

Airborne respiratory viruses are of particular concern because of the difficulty in controlling their transmission. Among these viruses, severe acute respiratory syndrome virus 2 (SARS-CoV-2), measles virus (MeV) and Henipavirus Nipah (NiV) and Hendra (HeV) can also infect the central nervous system (CNS) in humans and cause lethal encephalitis. For example, SARS-CoV-2, responsible for the COVID-19 pandemic, causes severe acute respiratory syndrome and neurological syndromes. Despite an effective vaccine, measles is reemerging and still responsible of more than 200 000 deaths per year. MeV can lead to measles inclusion-body encephalitis (MIBE) in immunocompromised patients or sub-acute sclerosing panencephalitis (SSPE) sometimes decades after exposure to the virus in immunocompetent patients. NiV and HeV are highly pathogenic zoonotic Paramyxoviruses that belong to the genus Henipavirus. Despite the low number of human cases recorded since their emergence in the late 1990s, NiV and HeV are classified among the top eight pathogens to prioritize for research and development in public health emergency contexts by the World Health Organization because of their high pandemic potential. Some strains are fatal in more than 70% of cases. To date, there is no effective commercialized treatment to cure these viral infections in human. Moreover, the early stages of the CNS infection by these three viruses remain poorly documented because most of the data come from post-mortem analyzes. The overall objective of this thesis was to identify factors influencing the CNS invasion by these viruses. The initial tropism, the dissemination, as well as the involvement of viral surface glycoproteins and viral genetic evolution were analyzed for SARS-CoV-2, MeV and several Henipavirus strains at the organic, cellular, and molecular levels. Two new models of organotypic cultures from hamster brainstem and lung have been developed and characterized. These ex vivo models are susceptible to the infection with SARS-CoV-2 and NiV. In contrast, a hyperfusogenic MeV mutant able to fuse in absence of known receptor, could only infect brain cultures. In these organotypic cultures the initial tropism of SARS-CoV-2 in the lungs was validated and the permissiveness of certain neurons in the brain was demonstrated. The results also showed that SARS-CoV-2 infection induces specific interferon and innate immune responses, along with cell death by apoptosis, necroptosis, and pyroptosis. Finally, these organotypic cultures have shown their relevance in validating the effect of antiviral treatments. The study of MeV carrying mutations in their fusion protein observed in measles encephalitis cases has shown the importance of the hyperfusogenic property to disseminate within the CNS, which lacks the expression of known receptors. Differences in the fusion machinery of three pathogenic Henipavirus strains were also identified and analyzed.Several antiviral candidates have been tested in organotypic brain cultures from hamsters and transgenic mice to block the dissemination of wild-type MeV and neuroinvasive MeV variants, but also of NiV and SARS-CoV-2. These results pave the way for the use of these ex vivo models to study newly emerged viruses’ pathogenesis and assess the efficacy of candidate antivirals before in vivo validation. The comparative study of the organotypic culture infections by these respiratory viruses with high pathogenicity differences illustrated how the fusion machinery can influence the viral dissemination in the brain.Les virus respiratoires aéroportés sont particulièrement préoccupants du fait de la difficulté de contrôler leur transmission. Parmi ces virus, le virus du syndrome respiratoire aigu sévère 2 (SARS-CoV-2), le virus de la rougeole (VR) et les Henipavirus Nipah (NiV) et Hendra (HeV) peuvent infecter également le système nerveux central (SNC) chez l’homme et provoquent alors souvent des encéphalites létales. Par exemple, le SARS-CoV-2, responsable de la pandémie de COVID-19, entraine un syndrome respiratoire aigu sévère et des atteintes neurologiques. De son coté, et malgré un vaccin efficace, la rougeole connait une réémergence inquiétante et cause la mort de plus de 200 000 personnes par an. Le VR peut entrainer des encéphalites rougeoleuses à corps d’inclusion (MIBE) dans un contexte d’immunodéficience ou une panencéphalite sclérosante subaiguë (PESS) parfois des décennies après l’exposition au virus chez des patients immunocompétents. NiV et HeV sont des Paramyxovirus zoonotiques hautement pathogènes du genre Henipavirus. Malgré le faible nombre de cas humains recensés depuis leur émergence à la fin des années 1990, les NiV et HeV sont classés parmi les huit pathogènes prioritaires pour la recherche par l’Organisation Mondiale de la Santé en raison de leur fort potentiel pandémique. Certaines souches sont mortelles dans plus de 70% des cas en moyenne. À ce jour il n’existe pas de traitement efficace commercialisé pour traiter ces infections virales chez l’homme. De plus, les étapes précoces de l’infection du SNC par ces virus restent peu documentées car la majorité des données proviennent d’analyses réalisées post mortem. L’objectif global de cette thèse a été d’identifier des facteurs influençant l’invasion du SNC par ces virus. Le tropisme initial, la dissémination, ainsi que l’implication des glycoprotéines virales de surface et l’évolution génétique virale ont été analysées pour le SARS-CoV-2, le VR et plusieurs souches d’Henipavirus à l’échelle organique, cellulaire et moléculaire. Deux nouveaux modèles de cultures organotypiques de poumons et de tronc cérébral chez le hamster ont été développés et caractérisés. Ces modèles ex vivo sont susceptibles à l’infection par le SARS-CoV-2 et par le NiV. En revanche, un mutant hyperfusogène du VR, pourtant capable de fusionner en l’absence de récepteur connu, n’infecte que les cultures de cerveau. Ces cultures organotypiques ont permis de valider le tropisme initial du SARS-CoV-2 dans les poumons et démontré la permissivité de certains neurones dans le cerveau. Ces modèles ont également permis d’établir que l’infection par le SARS-CoV-2 induit une réponse interféron spécifique et une réponse immunitaire innée, ainsi qu’une mort cellulaire par apoptose, nécroptose et pyroptose dans ces organes. Enfin, ces cultures organotypiques ont montré leur pertinence dans la validation de l’effet d’antiviraux. L’étude de VR portant des mutations dans leur protéine de fusion observées lors d’encéphalites rougeoleuses a montré l’importance du caractère hyperfusogène de ces mutants pour se disséminer dans le SNC pourtant dépourvu de récepteurs connus. Des différences dans la machinerie de fusion de trois souches pathogènes d’Henipavirus ont aussi été identifiées et analysées.Grace aux cultures organotypiques cérébrales de hamster et de souris transgéniques plusieurs candidats antiviraux ont été testés pour bloquer la dissémination du VR sauvage et de variants neuroinvasifs, mais aussi du NiV et du SARS-CoV-2. Ces résultats donnent des perspectives nouvelles d’utilisation de ces modèles ex vivo pour étudier l’infection par des virus émergents et pour évaluer l’efficacité de traitements en amont de validation in vivo. L’étude comparative de l’infection des cultures organotypiques par ces virus respiratoires à pathogénicité variable a illustré comment la machinerie de fusion peut influencer la dissémination virale dans le cerveau

Measles Encephalitis: Towards New Therapeutics

International audienceMeasles remains a major cause of morbidity and mortality worldwide among vaccine preventable diseases. Recent decline in vaccination coverage resulted in re-emergence of measles outbreaks. Measles virus (MeV) infection causes an acute systemic disease, associated in certain cases with central nervous system (CNS) infection leading to lethal neurological disease. Early following MeV infection some patients develop acute post-infectious measles encephalitis (APME), which is not associated with direct infection of the brain. MeV can also infect the CNS and cause sub-acute sclerosing panencephalitis (SSPE) in immunocompetent people or measles inclusion-body encephalitis (MIBE) in immunocompromised patients. To date, cellular and molecular mechanisms governing CNS invasion are still poorly understood. Moreover, the known MeV entry receptors are not expressed in the CNS and how MeV enters and spreads in the brain is not fully understood. Different antiviral treatments have been tested and validated in vitro, ex vivo and in vivo, mainly in small animal models. Most treatments have high efficacy at preventing infection but their effectiveness after CNS manifestations remains to be evaluated. This review describes MeV neural infection and current most advanced therapeutic approaches potentially applicable to treat MeV CNS infection

Interféron de type I et sélectivité de l’infection des cellules du système nerveux central par le virus de la rougeole

International audienceNo abstract availabl

Generation of Human and Equine cerebral organoids from iPSCs: tools to study neurotropic viruses

International audienceThe brain is a complex organ and any model for studying it in its normal and pathological aspects becomes a tool of choice for neuroscientists. The mastering and dissemination of protocols allowing brain organoids development have paved the way for a whole range of new studies in the field of brain development, modeling of neurodegenerative or neurodevelopmental diseases, understanding tumors as well as infectious diseases that affect the brain. While studies are so far limited to the use of human cerebral organoids (hOCs), there is a growing interest in having similar models in other species. First, by applying differentiation protocol to human induced pluripotent cells (hiPSc), we generate hOCs expressing early and late markers depending on differentiation time. Then, we success to induced them in ventral or dorsal forebrain revealed by two distinct specific gene expressions. The dorsal hOCs were successfully infected by neurotropic henipavirus and permits to highlight antiviral effect of molecules in brain model. Second, by applying somatic reprogramming protocol to equine primary cells, we generate equine induced pluripotent stem cells (eq-iPSc). These cells exhibit stem cell-like characteristics and show a plasticity to be induced in different embryonic lineages including ectoderm, precursor of neural tissue. The application of dorsal hOCs protocol on eq-iPSc allows the expression of (early and late) neural markers in equine embryoid bodies suggesting the generation of equine cerebral organoid (eq-OCs). These OCs represent an important experimental tool to study species-specific neurotropic viruses, screen antiviral molecules and also to perform inter-species comparation of zoonotic viruses in brain model

Generation of Human and Equine cerebral organoids from iPSCs: tools to study neurotropic viruses

International audienceThe brain is a complex organ and any model for studying it in its normal and pathological aspects becomes a tool of choice for neuroscientists. The mastering and dissemination of protocols allowing brain organoids development have paved the way for a whole range of new studies in the field of brain development, modeling of neurodegenerative or neurodevelopmental diseases, understanding tumors as well as infectious diseases that affect the brain. While studies are so far limited to the use of human cerebral organoids (hOCs), there is a growing interest in having similar models in other species. First, by applying differentiation protocol to human induced pluripotent cells (hiPSc), we generate hOCs expressing early and late markers depending on differentiation time. Then, we success to induced them in ventral or dorsal forebrain revealed by two distinct specific gene expressions. The dorsal hOCs were successfully infected by neurotropic henipavirus and permits to highlight antiviral effect of molecules in brain model. Second, by applying somatic reprogramming protocol to equine primary cells, we generate equine induced pluripotent stem cells (eq-iPSc). These cells exhibit stem cell-like characteristics and show a plasticity to be induced in different embryonic lineages including ectoderm, precursor of neural tissue. The application of dorsal hOCs protocol on eq-iPSc allows the expression of (early and late) neural markers in equine embryoid bodies suggesting the generation of equine cerebral organoid (eq-OCs). These OCs represent an important experimental tool to study species-specific neurotropic viruses, screen antiviral molecules and also to perform inter-species comparation of zoonotic viruses in brain model

Early Permissiveness of Central Nervous System Cells to Measles Virus Infection Is Determined by Hyperfusogenicity and Interferon Pressure

The cessation of measles virus (MeV) vaccination in more than 40 countries as a consequence of the COVID-19 pandemic is expected to significantly increase deaths due to measles. MeV can infect the central nervous system (CNS) and lead to lethal encephalitis. Substantial part of virus sequences recovered from patients’ brain were mutated in the matrix and/or the fusion protein (F). Mutations of the heptad repeat domain located in the C terminal (HRC) part of the F protein were often observed and were associated to hyperfusogenicity. These mutations promote brain invasion as a hallmark of neuroadaptation. Wild-type F allows entry into the brain, followed by limited spreading compared with the massive invasion observed for hyperfusogenic MeV. Taking advantage of our ex vivo models of hamster organotypic brain cultures, we investigated how the hyperfusogenic mutations in the F HRC domain modulate virus distribution in CNS cells. In this study, we also identified the dependence of neural cells susceptibility on both their activation state and destabilization of the virus F protein. Type I interferon (IFN-I) impaired mainly astrocytes and microglial cells permissiveness contrarily to neurons, opening a new way of consideration on the development of treatments against viral encephalitis

Measles fusion complexes from central nervous system clinical isolates: decreased interaction between hemagglutinin and fusion proteins

Abstract Measles virus (MeV) viral entry is mediated by a fusion complex comprised of a receptor binding protein (hemagglutinin, H) and a fusion protein (F). The wild type H/F complex requires interaction with specific proteinaceous receptors (CD150/SLAM and nectin-4) in order to be activated. In contrast the H/F complexes isolated from viruses infecting the central nervous system (CNS) do not require a specific receptor. A single amino acid change in the F protein (L454W) was previously identified in two patients with lethal sequelae of MeV CNS infection, and the F bearing this mutation mediates fusion even without the H protein. We show here that viruses bearing the L454W fusion complex are less efficient than wt virus at targeting receptor expressing cells and that this defect is associated with a decreased interaction between the H and the F proteins. Importance Measles (Mev) infection can cause serious complications including measles inclusion body encephalitis (MIBE) and subacute sclerosing panencephalitis (SSPE). MIBE and SSPE are relatively rare but lethal. We have shown that the fusion complex of CNS adapted clinical samples can spread in the absence of known receptor. We now provide evidence that HRC mutations leading to CNS adaptation come at a cost to the efficiency of viral entry. One Sentence Summary Measles CNS adapted fusion complexes have altered H/F interaction