Morphological and Molecular Changes in the Cortex and Cerebellum of Immunocompetent Mice Infected with Zika Virus.

Zika virus (ZIKV) disease continues to be a threat to public health, and it is estimated that millions of people have been infected and that there have been more cases of serious complications than those already reported. Despite many studies on the pathogenesis of ZIKV, several of the genes involved in the malformations associated with viral infection are still unknown. In this work, the morphological and molecular changes in the cortex and cerebellum of mice infected with ZIKV were evaluated. Neonatal BALB/c mice were inoculated with ZIKV intraperitoneally, and the respective controls were inoculated with a solution devoid of the virus. At day 10 postinoculation, the mice were euthanized to measure the expression of the markers involved in cortical and cerebellar neurodevelopment. The infected mice presented morphological changes accompanied by calcifications, as well as a decrease in most of the markers evaluated in the cortex and cerebellum. The modifications found could be predictive of astrocytosis, dendritic pathology, alterations in the regulation systems of neuronal excitation and inhibition, and premature maturation, conditions previously described in other models of ZIKV infection and microcephaly.

5'/3' RACE method for sequencing the 5' and 3' untranslated regions of Zika virus.

The spread of Zika virus (ZIKV) from the African continent to the Americas promoted its molecular evolution, as reflected by mutations in its RNA genome. Most of the ZIKV genome sequences in the GenBank database have incomplete 5' and 3' UTR sequences, reflecting the deficiency of whole-genome sequencing technologies to resolve the sequences of the genome ends. We modified a protocol for rapid amplification of cDNA ends (RACE) to determine the complete sequences of the 5' and 3' UTRs of a previously reported ZIKV isolate (GenBank no. MH544701.1). This strategy is useful for determining 5' and 3' UTR sequences of ZIKV isolates and will be useful for comparative genomics applications.

NeuN distribution in brain structures of normal and Zika-infected suckling mice.

Microcephaly is the more severe brain malformation because of Zika virus infection. Increased vulnerability of neural stem and progenitor cells to Zika infection during prenatal neurodevelopment impairs the complete formation of cortical layers. Normal development of cerebellum is also affected. However, the follow-up of apparently healthy children born to Zika exposed mothers during pregnancy has revealed other neurological sequelae. This suggests Zika infection susceptibility remains in nervous tissue after neurogenesis end, when differentiated neuronal populations predominate. The neuronal nuclear protein (NeuN) is an exclusive marker of postmitotic neurons. Changes in NeuN expression are associated with neuronal degeneration. We have evaluated immunohistochemical expression of NeuN protein in cerebral cortex, hippocampus, and cerebellum of normal and Zika-infected neonatal Balb/c mice. The highest NeuN immunoreactivity was found mainly in neurons of all cortical layers, pyramidal layer of hippocampus, granular layer of dentate gyrus and in internal granular layer of cerebellum. Viral infection caused marked loss of NeuN immunostaining in all these brain areas. This suggests neurodegenerative effects of Zika virus infection during postmitotic neuron maturation and contribute to interpretation of neuropathogenic mechanisms of Zika.

Novel Putative Tymoviridae-like Virus Isolated from Culex Mosquitoes in Colombia.

The family Tymoviridae comprises positive-sense RNA viruses, which mainly infect plants. Recently, a few Tymoviridae-like viruses have been found in mosquitoes, which feed on vertebrate sources. We describe a novel Tymoviridae-like virus, putatively named, Guachaca virus (GUAV), isolated from Culex pipiens and Culex quinquefasciatus species of mosquitoes and collected in the rural area of Santa Marta, Colombia. After a cytopathic effect was observed in C6/36 cells, RNA was extracted and processed through the NetoVIR next-generation sequencing protocol, and data were analyzed through the VirMAP pipeline. Molecular and phenotypic characterization of the GUAV was achieved using a 5'/3' RACE, transmission electron microscopy, amplification in vertebrate cells, and phylogenetic analysis. A cytopathic effect was observed in C6/36 cells three days post-infection. The GUAV genome was successfully assembled, and its polyadenylated 3' end was corroborated. GUAV shared only 54.9% amino acid identity with its closest relative, Ek Balam virus, and was grouped with the latter and other unclassified insect-associated tymoviruses in a phylogenetic analysis. GUAV is a new member of a family previously described as comprising plant-infecting viruses, which seem to infect and replicate in mosquitoes. The sugar- and blood-feeding behavior of the Culex spp., implies a sustained contact with plants and vertebrates and justifies further studies to unravel the ecological scenario for transmission.

Inmunohistochemical detection of pandemic SARSCoV- 2 antigens in lung tissue. / Detección inmunohistoquímica de antígenos de SARS-CoV-2 en tejido pulmonar

The COVID-19 pandemic caused by the SARS-CoV-2 virus has generated globally more than 110.7 million infections and 2.4 million deaths. The severity of this infection can range from asymptomatic, mild to severe. To know the possible associations between the presence of the virus and histopathological alterations found in tissues of fatal cases of COVID-19, the presence of the virus in the lung tissue of a patient with a clinical history of SARS-CoV-2 infection was evaluated. Lung tissue was histologically processed for immunohistochemical detection of SARSCoV-2. In the histopathological study, morphological changes associated with pneumonitis of viral origin were observed. Likewise, the location of the SARS-CoV-2 virus was observed mainly in the cytoplasm of the cells of the inflammatory infiltrate.

La pandemia de COVID-19 causada por el virus SARS-CoV-2 ha generado más de 110,7 millones de infecciones y 2,4 millones de muertes a nivel mundial. Esta infección puede ser asintomática y sus manifestaciones clínicas pueden variar entre leves y graves. Para conocer las posibles asociaciones entre la presencia del virus y las alteraciones histopatológicas encontradas en los tejidos de casos fatales de COVID-19, se evaluó la presencia del virus en el tejido pulmonar de un paciente con antecedentes clínicos de infección por SARS-CoV-2. La muestra se procesó para la detección inmunohistoquímica del virus. En el estudio histopatológico, se observaron cambios morfológicos asociados con neumonitis de origen viral. Asimismo, el virus se localizó principalmente en el citoplasma de las células del infiltrado inflamatorio.

Inmunohistochemical detection of pandemic SARS-CoV-2 antigens in lung tissue / Detección inmunohistoquímica de antígenos de SARS-CoV-2 en tejido pulmonar

The COVID-19 pandemic caused by the SARS-CoV-2 virus has generated globally more than 110.7 million infections and 2.4 million deaths. The severity of this infection can range from asymptomatic, mild to severe. To know the possible associations between the presence of the virus and histopathological alterations found in tissues of fatal cases of COVID-19, the presence of the virus in the lung tissue of a patient with a clinical history of SARS-CoV-2 infection was evaluated. Lung tissue was histologically processed for immunohistochemical detection of SARS- CoV-2. In the histopathological study, morphological changes associated with pneumonitis of viral origin were observed. Likewise, the location of the SARS-CoV-2 virus was observed mainly in the cytoplasm of the cells of the inflammatory infiltrate.

La pandemia de COVID-19 causada por el virus SARS-CoV-2 ha generado más de 110,7 millones de infecciones y 2,4 millones de muertes a nivel mundial. Esta infección puede ser asintomática y sus manifestaciones clínicas pueden variar entre leves y graves. Para conocer las posibles asociaciones entre la presencia del virus y las alteraciones histopatológicas encontradas en los tejidos de casos fatales de COVID-19, se evaluó la presencia del virus en el tejido pulmonar de un paciente con antecedentes clínicos de infección por SARS-CoV-2. La muestra se procesó para la detección inmunohistoquímica del virus. En el estudio histopatológico, se observaron cambios morfológicos asociados con neumonitis de origen viral. Asimismo, el virus se localizó principalmente en el citoplasma de las células del infiltrado inflamatorio.

CISH and IHC for the Simultaneous Detection of ZIKV RNA and Antigens in Formalin-Fixed Paraffin-Embedded Cell Blocks and Tissues.

Zika virus is an arthropod-borne virus that has recently emerged as a significant public health emergency due to its association with congenital malformations. Serological and molecular tests are typically used to confirm Zika virus infection. These methods, however, have limitations when the interest is in localizing the virus within the tissue and identifying the specific cell types involved in viral dissemination. Chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) are common histological techniques used for intracellular localization of RNA and protein expression, respectively. The combined use of CISH and IHC is important to obtain information about RNA replication and the location of infected target cells involved in Zika virus neuropathogenesis. There are no reports, however, of detailed procedures for the simultaneous detection of Zika virus RNA and proteins in formalin-fixed paraffin-embedded (FFPE) samples. Furthermore, the chromogenic detection methods for Zika virus RNA published thus far use expensive commercial kits, limiting their widespread use. As an alternative, we describe here a detailed and cost-effective step-by-step procedure for the simultaneous detection of Zika virus RNA and proteins in FFPE samples. First, we describe how to synthesize and purify homemade RNA probes conjugated with digoxygenin. Then, we outline the steps to perform the chromogenic detection of Zika virus RNA using these probes, and how to combine this technique with the immunodetection of viral antigens. To illustrate the entire workflow, we use FFPE samples derived from infected Vero cells as well as from human and mouse brain tissues. These methods are highly adaptable and can be used to study Zika virus or even other viruses of public health relevance, providing an optimal and economical alternative for laboratories with limited resources. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of RNA probes conjugated with digoxigenin (DIG) Basic Protocol 2: Simultaneous detection of ZIKV RNA and proteins in FFPE cell blocks and tissues.

An updated RT-qPCR assay for the simultaneous detection and quantification of chikungunya, dengue and zika viruses.

The real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for the detection and quantification of arboviruses, including Chikungunya, Dengue, and Zika viruses. In this study, an updated real-time RT-qPCR assay was designed and evaluated together with a synthetic positive-control chimeric RNA for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses. Amplification assays were performed to verify the construct integrity and optimal reaction/thermal cycling conditions. The analytical sensitivity of the assay was determined for each virus in single and multiplex reactions, as well as the performance in the detection and viral load quantification of experimental samples. The real-time RT-qPCR assay presented here allowed for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses and could be applied in several studies where the accurate quantification of viral genomes is required.

Comparative effect of osmium tetroxide and ruthenium tetroxide on Lacazia loboi ultrastructure.

Lobomycosis is a skin infection produced by the fungus Lacazia loboi, which mainly affects some indigenous and afro-descendant populations in Tropical America. We previously reported the comparative effect of osmium tetroxide (OsO4 ) and ruthenium tetroxide (RuO4 ) in the electron microscopy (EM) of other related microorganisms. The objective of this study is to compare the effect of postfixation with OsO4 and RuO4 in the ultrastructure of L. loboi yeasts. Skin biopsies on patients diagnosed with lobomycosis were fixed in glutaraldehyde at 3% and postfixed in the following solutions: (a) 1% OsO4 , (b) 0.2% RuO4 , and (c) OsO4 at 1% followed by RuO4 at 0.2%. They were then processed using the conventional method for EM. Unlike OsO4, the treatment with RuO4 revealed different shades of gray and electron dense bands in the cell wall and other cell components of L. loboi. The most notable finding was the presence of radial filamentous structures around the yeast, which made the image look like the sun. Postfixation with RuO4 revealed ultrastructural details that had not been previously reported for L loboi. The combined use of OsO4 and RuO4 in EM of microorganisms with cell walls can be useful to evaluate the effect of microbicide substances.

Effects of the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) on the nervous system. What can we expect from SARS -CoV-2? / [Efectos de los coronavirus del síndrome respiratorio agudo grave (SARS-CoV) y del síndrome respiratorio del Medio Oriente (MERS-CoV) en el sistema nervioso. ¿Qué esperar del SARS-CoV-2?]

Coronaviruses cause respiratory and gastrointestinal disorders in animals and humans. The current SARS-CoV-2, the COVID-19 infectious agent, belongs to a subgroup called betacoronavirus including the SARS-CoV and MERS-CoV responsible for epidemics in 2002 and 2012, respectively. These viruses can also infect the nervous system due to their affinity for the human angiotensin-converting enzyme 2 (ACE2) expressed in neurons and glial cells. Infections with SARS-CoV, MERS-CoV, and now SARS-CoV-2 also produce neurological signs such as acute cerebrovascular disease, impaired consciousness, and muscle injury, as well as dizziness, hypogeusia, hyposmia, hypoxia, neuralgia, and hypoxic encephalopathy. For this reason, close attention should be paid to the neurological manifestations of COVID-19 patients.

Los coronavirus son una familia de virus que se caracterizan por producir afectaciones respiratorias y gastrointestinales en animales y en seres humanos. El actual SARS-CoV-2, agente infeccioso de la COVID-19, pertenece a un subgrupo denominado betacoronavirus del que hacen parte el SARS-CoV y MERS-CoV, virus responsables de epidemias en el 2002 y el 2012, respectivamente. Estos virus también pueden infectar el sistema nervioso debido a su afinidad con la enzima convertidora de angiotensina humana 2 (ACE2), la cual se expresa en neuronas y células gliales. Se ha demostrado que las infecciones con SARS-CoV y MERS-CoV, y ahora también con el SARS-CoV-2, ocasionan condiciones neurológicas como la enfermedad cerebrovascular aguda, la conciencia alterada y las lesiones musculares, así como mareos, hipogeusia, hiposmia, hipoxia, neuralgia y encefalopatía hipóxica. Por ello debe prestarse mucha atención a las manifestaciones neurológicas de los pacientes de COVID-19.

Efectos de los coronavirus del síndrome respiratorio agudo grave (SARS-CoV) y del síndrome respiratorio del Medio Oriente (MERS-CoV) en el sistema nervioso. ¿Qué esperar del SARS-CoV-2? / Effects of the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) on the nervous system. What can we expect from SARS -CoV-2?

Los coronavirus son una familia de virus que se caracterizan por producir afectaciones respiratorias y gastrointestinales en animales y en seres humanos. El actual SARS-CoV-2, agente infeccioso de la COVID-19, pertenece a un subgrupo denominado betacoronavirus del que hacen parte el SARS-CoV y MERS-CoV, virus responsables de epidemias en el 2002 y el 2012, respectivamente. Estos virus también pueden infectar el sistema nervioso debido a su afinidad con la enzima convertidora de angiotensina humana 2 (ACE2), la cual se expresa en neuronas y células gliales. Se ha demostrado que las infecciones con SARS-CoV y MERS-CoV, y ahora también con el SARS-CoV-2, ocasionan condiciones neurológicas como la enfermedad cerebrovascular aguda, la conciencia alterada y las lesiones musculares, así como mareos, hipogeusia, hiposmia, hipoxia, neuralgia y encefalopatía hipóxica. Por ello debe prestarse mucha atención a las manifestaciones neurológicas de los pacientes de COVID-19.

Coronaviruses cause respiratory and gastrointestinal disorders in animals and humans. The current SARS-CoV-2, the COVID-19 infectious agent, belongs to a subgroup called betacoronavirus including the SARS-CoV and MERS-CoV responsible for epidemics in 2002 and 2012, respectively. These viruses can also infect the nervous system due to their affinity for the human angiotensin-converting enzyme 2 (ACE2) expressed in neurons and glial cells. Infections with SARS-CoV, MERS-CoV, and now SARS-CoV-2 also produce neurological signs such as acute cerebrovascular disease, impaired consciousness, and muscle injury, as well as dizziness, hypogeusia, hyposmia, hypoxia, neuralgia, and hypoxic encephalopathy. For this reason, close attention should be paid to the neurological manifestations of COVID-19 patients.

Effect of Postmortem Degradation on the Preservation of Viral Particles and Rabies Antigens in Mice Brains. Light and Electron Microscopic Study.

Rabies diagnosis is mainly made on fresh brain tissue postmortem by means of the direct immunofluorescence test. However, in some cases, it is not possible to use this technique, given that the affected nervous tissue goes through a postmortem degradation process, due to problems in the handling and transport of the samples. For this reason, the preservation in time of the rabies virus inclusions was assessed, as well as the immunoreactivity and the ultrastructure of viral particles in tissue with postmortem degradation. Brains of mice inoculated with rabies virus and control mice were processed for conventional histology, immunohistochemistry, electron microscopy, and immunoelectron microscopy in different postmortem times. In the processed tissues for hematoxylin and eosin, the presence of eosinophilic inclusions was not observed beyond 12 h postmortem. Surprisingly, the immunoreactivity of the viral antigens increased with time, at least until 30 h postmortem. It was possible to easily recognize the viral particles by means of conventional electron microscopy until 12 h postmortem. Immunoelectron microscopy allowed us to identify the presence of viral antigens disseminated in the neuronal cytoplasm until 30 h postmortem, but immunoreactive viral particles were not observed. The rabies infection did not cause histological or ultrastructural alterations different from those in the control group as a result of the postmortem degradation. In conclusion, the immunohistochemistry is a reliable test for rabies diagnosis in samples with postmortem degradation and that have been fixed with aldehydes.

Complete Genome Sequence of a Colombian Zika Virus Strain Obtained from BALB/c Mouse Brain after Intraperitoneal Inoculation.

A Zika virus (ZIKV) strain was isolated from an acute febrile patient during the Zika epidemics in Colombia. The strain was intraperitoneally inoculated into BALB/c mice, and 7 days postinoculation, neurological manifestations and ZIKV infection in the brain were demonstrated. The reported genome sequence is highly related to strains circulating in the Americas.

Neuroanatomical evidence of the transport of the rabies virus through the propriospinal tract in the spinal cord of mice

Introduction: Information about the neuroanatomical details of the ascendant transport of the rabies virus through the spinal cord is scarce. Objective: To identify the neuroanatomical route of dissemination of the rabies virus at each of the levels of the spinal cord of mice after being inoculated intramuscularly. Materials and methods: Mice were inoculated with the rabies virus in the hamstrings. After 24 hours post-inoculation, every eight hours, five animals were sacrificed by perfusion with paraformaldehyde. Then, the spinal cord was removed, and transverse cuts were made at the lumbosacral, thoracic, and cervical levels. These were processed by immunohistochemistry for the detection of viral antigens. Results: The first antigens of rabies were observed as aggregated particles in the lumbar spinal cord at 24 hours post-inoculation, within the ventral horn in the same side of the inoculated limb. At 32 hours post inoculation the first motoneurons immunoreactive to the virus became visible. At 40 hours postinoculation the first immunoreactive neurons were revealed in the thoracic level, located on lamina 8 and at 48 hours post-inoculation in the cervical cord, also on lamina 8. At 56 hours post-inoculation the virus had spread throughout the spinal cord, but the animals still did not show signs of the disease. Conclusion: In the mouse model we used, the rabies virus entered the spinal cord through the motoneurons and probably used the descending propriospinal pathway for its retrograde axonal transport to the encephalus.

Entry of rabies virus in the olfactory bulb of mice and effect of infection on cell markers of neurons and astrocytes / Ingreso del virus de la rabia en el bulbo olfatorio de ratones y efecto de la infección en marcadores celulares de neuronas y astrocitos

There are few studies of infection by rabies virus in the olfactory bulb (OB). This work was carried out with the purpose of establishing the time required to detect rabies antigens in the OB of mouse, after the intramuscular inoculation of the virus and to evaluate the effect of the infection on the expression of three proteins: calbindin (CB), parvalbumin (PV) and the glial fibrillary acidic protein (GFAP). Mice were inoculated with rabies virus intramuscularly in the hind limbs. Every 8 hours, after 72 hours postinoculation (p.i.), animals were sacrificed by perfusion with paraformaldehyde and coronal sections of OB were obtained for immunohistochemical study. These cuts were used to reveal the entry and spread of viral antigens. Tissue sections obtained in the terminal phase of the disease (144 hours p.i.), and controls of the same age were also processed for immunohistochemistry of CB, PV and GFAP. Rabies virus antigens were initially detected at 80 hours p.i. in a few mitral cells. At 88 hours p.i. the antigens had spread through most of these neurons but until the terminal phase of the disease there was little dispersion of the virus towards other cellular layers of the OB. The CB protein was expressed in cells of the glomerular stratum, the PV in cells of the outer plexiform layer and the GFAP was expressed in all the layers of the OB. Viral infection generated loss of CB expression and increase of PV expression. Immunoreactivity to GFAP was increased in the outer plexiform layer of the OB as a response to infection.

Son escasos los estudios de la infección por virus de la rabia en el bulbo olfatorio (OB). Este trabajo se realizó con el objetivo de establecer el tiempo requerido para detectar antígenos de rabia en el OB del ratón, luego de la inoculación intramuscular del virus y evaluar el efecto de la infección en la expresión de tres proteínas: calbindina (CB), parvoalbúmina (PV) y la proteína ácida fibrilar glial (GFAP). Los ratones fueron inoculados con virus de la rabia por vía intramuscular en las extremidades posteriores. Cada 8 horas, después de 72 horas de inoculación (p.i.), los animales se sacrificaron por perfusión con paraformaldehído y se obtuvieron secciones coronales de OB para el estudio inmunohistoquímico. Estos cortes se usaron para revelar la entrada y propagación de antígenos virales. Las secciones de tejido obtenidas en la fase terminal de la enfermedad (144 horas p.i.), y los controles de la misma edad también se procesaron para inmunohistoquímica de CB, PV y GFAP. Los antígenos del virus de la rabia se detectaron inicialmente a las 80 horas p.i. en unas pocas células mitrales. A las 88 horas p.i. los antígenos se habían diseminado a través de la mayoría de estas neuronas, pero hasta la fase terminal de la enfermedad había poca dispersión del virus hacia otras capas celulares del OB. La proteína CB se expresó en las células del estrato glomerular, la PV en células de la capa plexiforme externa y la GFAP se expresó en todas las capas del OB. La infección viral generó pérdida de expresión de CB y aumento en la expresión de PV. La inmunorreactividad a GFAP aumentó en la capa plexiforme externa del OB como respuesta a la infección.

Evidencia neuroanatómica del transporte del virus de la rabia por la vía propioespinal de la médula espinal de ratones / Neuroanatomical evidence of the transport of the rabies virus through the propriospinal tract in the spinal cord of mice

Resumen Introducción. Es escasa la información sobre los detalles neuroanatómicos del transporte del virus de la rabia en su ascenso por la médula espinal. Objetivos. Identificar la ruta neuroanatómica de diseminación del virus de la rabia en cada uno de los niveles de la médula espinal de ratón, después de ser inoculado por vía intramuscular. Materiales y métodos. Se inocularon ratones en los músculos isquiotibiales, con virus de la rabia. A partir de las 24 horas después de la inoculación, cada ocho horas se sacrificaron cinco animales por perfusión con paraformaldehído, se les extrajo la médula espinal y se hicieron cortes transversales en los niveles lumbosacro, torácico y cervical. Estos se procesaron mediante inmunohistoquímica para detectar antígenos virales. Resultados. Los primeros antígenos de la rabia se observaron como partículas agregadas, en la médula espinal lumbar, a las 24 horas después de la inoculación, dentro del asta ventral ipsilateral a la extremidad inoculada. A las 32 horas después de la inoculación, se hicieron visibles las primeras motoneuronas inmunorreactivas al virus. A las 40 horas después de la inoculación, se revelaron las primeras neuronas inmunorreactivas en la médula torácica, localizadas en la lámina 8 y, a las 48 horas después de la inoculación en la médula cervical, también en la lámina 8. A las 56 horas después de la inoculación, el virus se había diseminado por toda la médula espinal pero los animales aún no revelaban signos de la enfermedad. Conclusión. En el modelo de ratón aquí utilizado, el virus de la rabia ingresó a la médula espinal por las motoneuronas y, probablemente, utilizó la vía propioespinal descendente para su transporte axonal retrógrado hasta el encéfalo.

Abstract Introduction: Information about the neuroanatomical details of the ascendant transport of the rabies virus through the spinal cord is scarce. Objective: To identify the neuroanatomical route of dissemination of the rabies virus at each of the levels of the spinal cord of mice after being inoculated intramuscularly. Materials and methods: Mice were inoculated with the rabies virus in the hamstrings. After 24 hours post-inoculation, every eight hours, five animals were sacrificed by perfusion with paraformaldehyde. Then, the spinal cord was removed, and transverse cuts were made at the lumbosacral, thoracic, and cervical levels. These were processed by immunohistochemistry for the detection of viral antigens. Results: The first antigens of rabies were observed as aggregated particles in the lumbar spinal cord at 24 hours post-inoculation, within the ventral horn in the same side of the inoculated limb. At 32 hours post inoculation the first motoneurons immunoreactive to the virus became visible. At 40 hours post-inoculation the first immunoreactive neurons were revealed in the thoracic level, located on lamina 8 and at 48 hours post-inoculation in the cervical cord, also on lamina 8. At 56 hours post-inoculation the virus had spread throughout the spinal cord, but the animals still did not show signs of the disease. Conclusion: In the mouse model we used, the rabies virus entered the spinal cord through the motoneurons and probably used the descending propriospinal pathway for its retrograde axonal transport to the encephalus.