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Yves Gaudin - One of the best experts on this subject based on the ideXlab platform.
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Negri Bodies and other virus membrane-less replication compartments
Biochimica et Biophysica Acta - Molecular Cell Research, 2020Co-Authors: Quentin Nevers, Cécile Lagaudrière-gesbert, Aurélie A. Albertini, Yves GaudinAbstract:Viruses reshape the organization of the cell interior to achieve different steps of their cellular cycle. Particularly, viral replication and assembly often take place in viral factories where specific viral and cellular proteins as well as nucleic acids concentrate. Viral factories can be either membrane-delimited or devoid of any cellular membranes. In the latter case, they are referred as membrane-less replication compartments. The most emblematic ones are the Negri Bodies, which are inclusion Bodies that constitute the hallmark of rabies virus infection. Interestingly, Negri Bodies and several other viral replication compartments have been shown to arise from a liquid-liquid phase separation process and, thus, constitute a new class of liquid organelles. This is a paradigm shift in the field of virus replication. Here, we review the different aspects of membrane-less virus replication compartments with a focus on the Mononegavirales order and discuss their interactions with the host cell machineries and the cytoskeleton. We particularly examine the interplay between viral factories and the cellular innate immune response, of which several components also form membrane-less condensates in infected cells.
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Structure and Function of Negri Bodies
Advances in experimental medicine and biology, 2019Co-Authors: Jovan Nikolic, Danielle Blondel, Nathalie Scrima, Cécile Lagaudrière-gesbert, Yves GaudinAbstract:Replication and assembly of many viruses occur in viral factories which are specialized intracellular compartments formed during viral infection. For rabies virus, those viral factories are called Negri Bodies (NBs). NBs are cytoplasmic inclusion Bodies in which viral RNAs (mRNAs as well as genomic and antigenomic RNAs) are synthesized. NBs are spherical, they can fuse together, and can reversibly deform when encountering a physical barrier. All these characteristics are similar to those of eukaryotic membrane-less liquid organelles which contribute to the compartmentalization of the cell interior. Indeed, the liquid nature of NBs has been confirmed by FRAP experiments. The co-expression of rabies virus nucleoprotein N and phosphoprotein P is sufficient to induce the formation of cytoplasmic inclusions recapitulating NBs properties. Remarkably, P and N have features similar to those of cellular proteins involved in liquid organelles formation: N is an RNA-binding protein and P contains intrinsically disordered domains. An overview of the literature indicates that formation of liquid viral factories by phase separation is probably common among Mononegavirales. This allows specific recruitment and concentration of viral proteins. Finally, as virus-associated molecular patterns recognized by cellular sensors of RNA virus replication are probably essentially present in the viral factory, there should be a subtle interplay (which remains to be characterized) between those liquid structures and the cellular proteins which trigger the innate immune response.
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Negri Bodies are viral factories with properties of liquid organelles
Nature Communications, 2017Co-Authors: Jovan Nikolic, Yves Gaudin, Romain Le Bars, Zoé Lama, Nathalie Scrima, Cécile Lagaudrière-gesbert, Danielle BlondelAbstract:Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri Bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri Bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.Negative strand RNA viruses, such as rabies virus, induce formation of cytoplasmic inclusions for genome replication. Here, Nikolic et al. show that these so-called Negri Bodies (NBs) have characteristics of liquid organelles and they identify the minimal protein domains required for NB formation.
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functional characterization of Negri Bodies nbs in rabies virus infected cells evidence that nbs are sites of viral transcription and replication
Journal of Virology, 2009Co-Authors: Xavier Lahaye, Aurore Vidy, Carole Pomier, Linda Obiang, Francis Harper, Yves Gaudin, Danielle BlondelAbstract:Rabies virus infection induces the formation of cytoplasmic inclusion Bodies that resemble Negri Bodies found in the cytoplasm of some infected nerve cells. We have studied the morphogenesis and the role of these Negri body-like structures (NBLs) during viral infection. The results indicate that these spherical structures (one or two per cell in the initial stage of infection), composed of the viral N and P proteins, grow during the virus cycle before appearing as smaller structures at late stages of infection. We have shown that the microtubule network is not necessary for the formation of these inclusion Bodies but is involved in their dynamics. In contrast, the actin network does not play any detectable role in these processes. These inclusion Bodies contain Hsp70 and ubiquitinylated proteins, but they are not misfolded protein aggregates. NBLs, in fact, appear to be functional structures involved in the viral life cycle. Specifically, using in situ fluorescent hybridization techniques, we show that all viral RNAs (genome, antigenome, and every mRNA) are located inside the inclusion Bodies. Significantly, short-term RNA labeling in the presence of BrUTP strongly suggests that the NBLs are the sites where viral transcription and replication take place.
Monique Lafon - One of the best experts on this subject based on the ideXlab platform.
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Toll-like receptor 3 (tlr3) plays a major role in the formation of rabies virus Negri Bodies. PLoS Pathog
2016Co-Authors: Pascal Roux, Annemarie Le Sourd, Mireille Lafage, Anne Danckaert, Jeanpierre Bourgeois, Monique LafonAbstract:Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion Bodies. As well as TLR3, these inclusion Bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion Bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR32/2 mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit
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toll like receptor 3 tlr3 plays a major role in the formation of rabies virus Negri Bodies
PLOS Pathogens, 2009Co-Authors: Pauline Menager, Francoise Megret, Annemarie Le Sourd, Mireille Lafage, Christophe Prehaud, Pascal Roux, Anne Danckaert, Jeanpierre Bourgeois, Monique LafonAbstract:Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion Bodies. As well as TLR3, these inclusion Bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion Bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.
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Toll-like receptor 3 (TLR3) plays a major role in the formation of rabies virus Negri Bodies. PLoS Pathog 5
2009Co-Authors: Pauline Menager, Francoise Megret, Annemarie Le Sourd, Mireille Lafage, Christophe Prehaud, Pascal Roux, Anne Danckaert, Jeanpierre Bourgeois, Monique LafonAbstract:Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion Bodies. As well as TLR3, these inclusion Bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion Bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3 2/2 mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. Thi
Claudio S.l. Barros - One of the best experts on this subject based on the ideXlab platform.
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Ocorrência de raiva em ovinos no Rio Grande do Sul Occurrence of rabies in sheep in Rio Grande do Sul, Brazil
Colégio Brasileiro de Patologia Animal (CBPA), 2008Co-Authors: Daniel R. Rissi, Felipe Pierezan, Glaucia D. Kommers, Claudio S.l. BarrosAbstract:Descreve-se a ocorrência de raiva em ovinos na região Central do Rio Grande do Sul em novembro de 2003. Foram afetados dois ovinos de raça mista, um macho de três meses e uma fêmea de 2,5 anos de idade que apresentaram sinais clínicos com evolução de cinco dias e caracterizados por dificuldade de locomoção, tremores musculares, decúbito lateral, convulsões, opistótono e febre. Histologicamente havia mielomeningoencefalite não-supurativa, associada a inclusões eosinofílicas intracitoplasmáticas (corpúsculos de Negri) em neurônios nos dois ovinos afetados. Em um ovino em que o gânglio de Gasser foi examinado, havia ganglionite não-supurativa. As lesões concentravam-se predominantemente na substância cinzenta da medula espinhal, no tronco encefálico e no cerebelo. Antígeno viral foi detectado em seções selecionadas de ponte e bulbo submetidas ao teste de imuno-histoquímica utilizando anticorpo policlonal anti-ribonucleoproteína do vírus da raiva. Os casos ocorreram em meio a um surto de raiva bovina transmitida por morcegos e foram considerados, com bases epidemiológicas, como transmitidos da mesma forma, como ocorre na raiva endêmica de bovinos no Rio Grande do Sul.Cases of rabies in sheep occurring in November 2003, in central Rio Grande do Sul state, Brazil, are described. A 3-month-old male, and a 2.5-month-old female sheep were affected. Clinical signs were characterized by abnormal gait, trembling, lateral recumbency, convulsion, opisthotonus, and fever. Histological findings included a non-suppurative myelomeningoencephalitis associated with intracytoplasmic eosinophilic inclusion (Negri) Bodies in neurons. Lesions were predominantly observed in gray matter of the spinal cord, brainstem and cerebellum. There was non-suppurative Gasserian ganglionitis in one sheep in which this structure was examined. Immunohistochemistry using rabies virus ribonucleoprotein polyclonal antibody yelded positive result in brain tissue from both sheep. The cases herein reported occurred concomitantly to an outbreak of bat transmitted rabies in cattle in the same region and were considered, on epidemiological basis, to be transmitted by bats, as occurs in endemic bovine rabies in Rio Grande do Sul
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Histomorfologia do gânglio de Gasser, da rete mirabile carotídea e da hipófise de bovinos: estudo de 199 casos
Pesquisa Veterinária Brasileira, 2006Co-Authors: Raquel R. Rech, Daniel R. Rissi, Marcia Cristina Da Silva, Maria Andréia Inkelmann, Claudio S.l. BarrosAbstract:The complex of heterogenous tissues formed by the Gasserian ganglia, carotid rete mirabile and pituitary gland (GRH) from 199 cattle were grossly and histologically evaluated. Twenty one of the GRH were from comfirmed cases of rabies, 19 were diagnosed as cases of meningoencephalitis caused by bovine herpesvirus-5 (BoHV-5), 11 were confirmed cases of malignant catarrhal fever (MCF), 7 had pituitary abscesses, 17 had lesions of hepatic encephalopathy (status spongiosus) attributable to chronic liver failure due to Senecio sp poisoning; and the remaining 124 GRH were from cattle which did not present neurological signs and died or were euthanatized due to various causes. No significant histologic changes were observed in the GRH from the 124 cattle which did not present neurological signs, in the GRH of the 17 cattle that had hepatic encephalopathy neither in the GRH of the 19 cattle diagnosed with BoHV-5 meningoencephalitis. Inflammatory changes were found in the Gasserian ganglia of 20 out of 21 cases of rabies and consisted of proliferation of sattelite cells, glial nodules and lymphoplasmacytic infiltrate; degenerative changes included central chromatolysis, neuronal vacuolation, and neuronal death with neuronophagia. Characteristic eosinophilic intracytoplasmatic inclusion (Negri) Bodies were found in 19 out of 21 cases of rabies, neuritis of the fifth nerve in 11, and neurohypophysitis in 4 cases. The GRH from nine of the eleven cases of MCF had characteristic necrotizing arteritis affecting the arteries of the carotid rete mirabile. In seven cases pituitary abscesses were found; they were characterized by large aggregates of neutrophils admixed with cellular debris and surrounded by mononuclear cells and were walled by a fibrous capsule. Several incidental findings were observed equally in the 199 histologically examined GRH and consequently were non specific for any disease entity. These include cysts in the pituitary cleft, fibrin and neutrophilic aggregates in the cavernous sinus of the carotid rete mirabile, mildly increased numbers of sattelite cells around neurons of the Gasserian ganglia and occasional neuronal vacuolation and lipofuscinosis. It was concluded that the histological examination of the GRH is an important and reliable tool in the diagnosis of the main forms of encephalitis occurring in cattle in Brazil.
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Histomorfologia do gânglio de Gasser, da rete mirabile carotídea e da hipófise de bovinos: estudo de 199 casos Histomorphology of the Gasserian ganglion, carotid rete mirabile and pituitary gland in cattle: a study of 199 cases
Colégio Brasileiro de Patologia Animal (CBPA), 2006Co-Authors: Raquel R. Rech, Daniel R. Rissi, Marcia Cristina Da Silva, Maria Andréia Inkelmann, Claudio S.l. BarrosAbstract:O complexo heterogêneo de tecidos formado pelo gânglio de Gasser, rete mirabile carotídea e hipófise (GRH) de 199 bovinos foram macro e microscopicamente avaliados. Vinte e um GRH eram de casos confirmados de raiva, 19 com diagnóstico de meningoencefalite por herpesvírus bovino-5 (BoHV-5), 11 casos de febre catarral maligna (FCM), 7 tinham abscesso de pituitária, 17 apresentaram lesões de encefalopatia hepática (status spongiosus) atribuído à fibrose crônica por intoxicação por Senecio sp; e os 124 GRH restantes eram provenientes de bovinos que não apresentaram sinais neurológicos e que morreram ou foram eutanasiados por causas diversas. Nenhuma alteração histológica significativa foi observada no GRH dos 124 bovinos que não tinham sinais neurológicos, nos 17 bovinos com encefalopatia hepática, nem nos 19 bovinos afetados por meningoencefalite por BoHV-5. Alterações inflamatórias foram encontradas em 20 dos 21 casos de raiva e consistiam de proliferação de células satélites, nódulos gliais e infiltrado linfo-plasmocitário; alterações degenerativas incluíam cromatólise central, vacuolização neuronal e necrose neuronal com neuronofagia. Corpúsculos de inclusão eosinofílicos intracitoplasmáticos (de Negri) foram encontrados em 19 dos 21 casos de raiva; neurite do nervo trigêmeo em 11 casos e neuroipofisite em 4 casos. O complexo GRH de 9 de 11 casos de FCM apresentava arterite necrosante, que afetava as artérias da rete mirabile carotídea. Em 7 dos 199 GRH examinados havia abscessos de pituitária caracterizados por agregados de neutrófilos e detritos celulares circundados por infiltrado mononuclear e cápsula fibrosa. Vários achados incidentais foram observados nos 199 GRH examinados histologicamente e não foram correlacionados com alterações ligadas a doenças. Estes incluíam cistos na cavidade hipofisária, agregados de fibrina e neutrófilos no seio cavernoso da rete mirabile carotídea, leve aumento do número de células satélites ao redor dos neurônios do gânglio de Gasser e ocasional vacuolização e lipofuscinose neuronal. O exame histológico do complexo GRH é uma ferramenta importante e confiável no diagnóstico das principais encefalites em bovinos no Brasil.The complex of heterogenous tissues formed by the Gasserian ganglia, carotid rete mirabile and pituitary gland (GRH) from 199 cattle were grossly and histologically evaluated. Twenty one of the GRH were from comfirmed cases of rabies, 19 were diagnosed as cases of meningoencephalitis caused by bovine herpesvirus-5 (BoHV-5), 11 were confirmed cases of malignant catarrhal fever (MCF), 7 had pituitary abscesses, 17 had lesions of hepatic encephalopathy (status spongiosus) attributable to chronic liver failure due to Senecio sp poisoning; and the remaining 124 GRH were from cattle which did not present neurological signs and died or were euthanatized due to various causes. No significant histologic changes were observed in the GRH from the 124 cattle which did not present neurological signs, in the GRH of the 17 cattle that had hepatic encephalopathy neither in the GRH of the 19 cattle diagnosed with BoHV-5 meningoencephalitis. Inflammatory changes were found in the Gasserian ganglia of 20 out of 21 cases of rabies and consisted of proliferation of sattelite cells, glial nodules and lymphoplasmacytic infiltrate; degenerative changes included central chromatolysis, neuronal vacuolation, and neuronal death with neuronophagia. Characteristic eosinophilic intracytoplasmatic inclusion (Negri) Bodies were found in 19 out of 21 cases of rabies, neuritis of the fifth nerve in 11, and neurohypophysitis in 4 cases. The GRH from nine of the eleven cases of MCF had characteristic necrotizing arteritis affecting the arteries of the carotid rete mirabile. In seven cases pituitary abscesses were found; they were characterized by large aggregates of neutrophils admixed with cellular debris and surrounded by mononuclear cells and were walled by a fibrous capsule. Several incidental findings were observed equally in the 199 histologically examined GRH and consequently were non specific for any disease entity. These include cysts in the pituitary cleft, fibrin and neutrophilic aggregates in the cavernous sinus of the carotid rete mirabile, mildly increased numbers of sattelite cells around neurons of the Gasserian ganglia and occasional neuronal vacuolation and lipofuscinosis. It was concluded that the histological examination of the GRH is an important and reliable tool in the diagnosis of the main forms of encephalitis occurring in cattle in Brazil
Renato Augusto Da ,silva - One of the best experts on this subject based on the ideXlab platform.
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Ensaios com o vírus rábico "ERA", no Brasil. I. Distribuição do vírus no organismo de bovinos inoculados e formação de inclusões no sistema nervoso central
Embrapa, 2014Co-Authors: Renato Augusto Da ,silva, Norma Moraes Da ,silva, Menezes, Patino Roberto ValadãoAbstract:Rabies virus, "ERA" strain, inoculated by intra-muscular route into the masseter muscle was capable of localizing in the salivary glands, brain, cerebellum, bulbus and spinal cord. The "ERA" strain determined the appearence of inclusions, in the cytoplasm of the neurons of bulbus and spinal cord, which resembled Negri Bodies. Fine granular acidophile material diffusely dispersed in the cytoplasm was encountered. The viral rabies antigen was detected in high concentration in the bulbus, spinal cord and salivary glands, by fluorescent antiBodies techniques. The rabies virus was not recovered from the nervous tissues, salivary glands saliva of mice.A amostra de vírus rábido ERA, inoculada por via intramuscular em bovinos, no masseter, foi capaz de se localizar nos tecidos das glândulas salivares (submaxilares, parótidas e sublinguais) e no sistema nervoso central. A amostra ERA determinou o aparecimento de inclusões no citoplasma e nos prolongamentos dos neurônios dos bovinos, muito semelhantes aos corpúsculos de Negri. Material acidófilo finamente granular, espalhado difusamente no citoplasma celular, pôde ser observado. Os camundongos que receberam intracranialmente a amostra ERA também mostraram inclusões muito semelhantes aos corpúsculos de Negri no citoplasma de suas células nervosas. O antígeno rábico nos tecidos glandular e nervoso foi evidenciado pelo teste de anticorpos fluorescentes. Não se recuperou vírus dos tecidos imuno-positivos, nem da saliva, por inoculação em camundongos adultos e lactentes
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Infecção rábica em cão com presença de vírus virulento nas glândulas salivares e avirulência no encéfalo
'Embrapa Agroindustria Tropical', 2014Co-Authors: Renato Augusto Da ,silva, Norma Moraes Da ,silvaAbstract:This paper reports a case study of rabies in a domestic dog. Diagnoses of rabies were made in the laboratory by the fluorescent antibody technique, the detection of Negri Bodies and mouse innoculation. The presence of the rabies viral antigen in the brain, cerebellum and salivary glands of the dog was shown by the fluorescent antibody technique. The rabies virus was isolated from the salivary glands but not from the central nervous system, by the mouse inoculation technique.É relatado um caso de raiva em cão, diagnosticado laboratorialmente pelos métodos de imunoflorescência, pesquisa de corpúsculos de Negri pela técnica de Faraco e inoculação em camundongos. Constatou-se a presença de antígeno viral rábico no cérebro, cerebelo e glândulas submaxilares, não se isolando vírus do encéfalo e sim do tecido glandular, apesar de estar o vírus presente nos tecidos nervosos, conforme visualização à microscopia fluorescente
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Isolamento do vírus rábico de morcêgo insetívoro, Histiotus velatus, capturado no Estado de Santa Catarina
'Embrapa Agroindustria Tropical', 2014Co-Authors: Adalvio Fernandes De ,amorim, Renato Augusto Da ,silva, Norma Moraes Da ,silvaAbstract:Rabies virus was isolated from the brain of an insectivorous bat, Histiotus velatus Geoffroy, captured in the Municipality of Alfredo Wagner, State of Santa Catarina (South Brazil). The brain suspension was inoculated into adult mice by intracerebral route. The virus strain was identified as rabies virus by the presence of Negri Bodies in the cytoplasm of nervous cells from brains of mice inoculated at 21 days of age. Identification was made by neutralization test using a standard rabies immune serum and by fluorescent antibody technique.Isolou-se vírus rábico do cérebro de morcêgo insetívoro, Histiotus velatus Geoffroy, capturado no Município de Alfredo Wagner, Estado de Santa Catarina. A suspensão do cérebro foi inoculada em camundongos de 21 dias de idade por via intracraniana, sendo a amostra de vírus identificada como vírus rábico pelos sintomas apresentados pelos camundongos, formação de corpúsculos de Negri, prova de sòro-neutralização e técnica de imunofluorescência
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Ocorrência da raiva em lobo-guará (Chrysocyon brachyurus, Illiger 1815)
'Embrapa Agroindustria Tropical', 2014Co-Authors: Renato Augusto Da ,silva, Breckenfeld, Silvio Guilherme B.Abstract:Rabies virus was isolated from brain and submaxillary gland of three wolves in conditions of captivity. The occurrence of rabies was determined by the appearance of Negri Bodies in the brains of the wolves and verified by intercerebral inoculation of adult mice. One of the samples of virus was neutralized by an anti-rabies serum of known neutralizing capability.O vírus da raiva foi isolado do cérebro de três lobos-guarás (Chrysocyon brachyurus) e da glândula submaxilar de um deles, inoculando-se camundongos adultos por via intracraniana com as suspensões destes tecidos. Os guarás encontravam-se em regime de cativeiro no Zoobotânico de Brasília há mais de um ano. As amostras de vírus determinaram o aparecimento de corpúsculos de Negri nos cérebros dos lobos, como também, nos cérebros dos camundongos inoculados. Uma destas amostras de vírus foi neutralizada por um soro anti-rábico de reconhecida capacidade neutralizante. A presença do vírus rábico em roedores no Zoobotânico não foi constatada
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Isolamento de vírus rábico do rim, coração e cérebro de bovino na doença natural
'Embrapa Agroindustria Tropical', 2014Co-Authors: Renato Augusto Da ,silva, Ary Moreira De ,souza, Lima, Altair CorrêaAbstract:Rabies virus was isolated by intracerebral inoculation of baby and adult mice by using emulsions from kidney, heart and brain tissues of a naturally infected cow in the County of São Fidelis, State of Rio de Janeiro, Brazil. The virus strains isolated from different tissues were identified as rabies virus by microscopic examination of the brains of inoculated mice, revealing presence of Negri Bodies in the citoplasm of nervous cells and by serum neutralization tests using standardized rabies serum.De uma vaca mestiça zebu, acometida de raiva, no Município de São Fidelis no Estado do Rio de Janeiro, foi isolado vírus da raiva do rim, coração e cérebro, por inoculação intracerebral das emulsões destes tecidos, em camundongos lactentes (4 dias de idade) e adultos (21 dias de idade). As amostras de vírus, isoladas dos diferentes tecidos, foram identificadas como vírus rábico pela formação de corpúsculos de Negri no citoplasma das células nervosas dos cérebros dos camundongos inoculados e pela prova de soro-neutralização, neutralizando-se para esta prova um soro anti-rábico de reconhecida capacidade neutralizante
Danielle Blondel - One of the best experts on this subject based on the ideXlab platform.
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Structure and Function of Negri Bodies
Advances in experimental medicine and biology, 2019Co-Authors: Jovan Nikolic, Danielle Blondel, Nathalie Scrima, Cécile Lagaudrière-gesbert, Yves GaudinAbstract:Replication and assembly of many viruses occur in viral factories which are specialized intracellular compartments formed during viral infection. For rabies virus, those viral factories are called Negri Bodies (NBs). NBs are cytoplasmic inclusion Bodies in which viral RNAs (mRNAs as well as genomic and antigenomic RNAs) are synthesized. NBs are spherical, they can fuse together, and can reversibly deform when encountering a physical barrier. All these characteristics are similar to those of eukaryotic membrane-less liquid organelles which contribute to the compartmentalization of the cell interior. Indeed, the liquid nature of NBs has been confirmed by FRAP experiments. The co-expression of rabies virus nucleoprotein N and phosphoprotein P is sufficient to induce the formation of cytoplasmic inclusions recapitulating NBs properties. Remarkably, P and N have features similar to those of cellular proteins involved in liquid organelles formation: N is an RNA-binding protein and P contains intrinsically disordered domains. An overview of the literature indicates that formation of liquid viral factories by phase separation is probably common among Mononegavirales. This allows specific recruitment and concentration of viral proteins. Finally, as virus-associated molecular patterns recognized by cellular sensors of RNA virus replication are probably essentially present in the viral factory, there should be a subtle interplay (which remains to be characterized) between those liquid structures and the cellular proteins which trigger the innate immune response.
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Negri Bodies are viral factories with properties of liquid organelles
Nature Communications, 2017Co-Authors: Jovan Nikolic, Yves Gaudin, Romain Le Bars, Zoé Lama, Nathalie Scrima, Cécile Lagaudrière-gesbert, Danielle BlondelAbstract:Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri Bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri Bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.Negative strand RNA viruses, such as rabies virus, induce formation of cytoplasmic inclusions for genome replication. Here, Nikolic et al. show that these so-called Negri Bodies (NBs) have characteristics of liquid organelles and they identify the minimal protein domains required for NB formation.
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Rabies Virus Infection Induces the Formation of Stress Granules Closely Connected to the Viral Factories
PLoS Pathogens, 2016Co-Authors: Jovan Nikolic, Zoé Lama, Cécile Lagaudrière-gesbert, Ahmet Civas, Danielle BlondelAbstract:Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri Bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection.
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focal adhesion kinase is involved in rabies virus infection through its interaction with viral phosphoprotein p
Journal of Virology, 2015Co-Authors: Baptiste Fouquet, Jovan Nikolic, Hervé Bourhy, Florence Larrous, Christoph Wirblich, Cecile Lagaudrieregesbert, Danielle BlondelAbstract:The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri Bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function.
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functional characterization of Negri Bodies nbs in rabies virus infected cells evidence that nbs are sites of viral transcription and replication
Journal of Virology, 2009Co-Authors: Xavier Lahaye, Aurore Vidy, Carole Pomier, Linda Obiang, Francis Harper, Yves Gaudin, Danielle BlondelAbstract:Rabies virus infection induces the formation of cytoplasmic inclusion Bodies that resemble Negri Bodies found in the cytoplasm of some infected nerve cells. We have studied the morphogenesis and the role of these Negri body-like structures (NBLs) during viral infection. The results indicate that these spherical structures (one or two per cell in the initial stage of infection), composed of the viral N and P proteins, grow during the virus cycle before appearing as smaller structures at late stages of infection. We have shown that the microtubule network is not necessary for the formation of these inclusion Bodies but is involved in their dynamics. In contrast, the actin network does not play any detectable role in these processes. These inclusion Bodies contain Hsp70 and ubiquitinylated proteins, but they are not misfolded protein aggregates. NBLs, in fact, appear to be functional structures involved in the viral life cycle. Specifically, using in situ fluorescent hybridization techniques, we show that all viral RNAs (genome, antigenome, and every mRNA) are located inside the inclusion Bodies. Significantly, short-term RNA labeling in the presence of BrUTP strongly suggests that the NBLs are the sites where viral transcription and replication take place.
