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.

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.

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.

Characterization of the emerging B.1.621 variant of interest of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity has the potential to impact the virus transmissibility and the escape from natural infection- or vaccine-elicited neutralizing antibodies. Here, representative samples from circulating SARS-CoV-2 in Colombia between January and April 2021, were processed for genome sequencing and lineage determination following the nanopore amplicon ARTIC network protocol and PANGOLIN pipeline. This strategy allowed us to identify the emergence of the B.1.621 lineage, considered a variant of interest (VOI) with the accumulation of several substitutions affecting the Spike protein, including the amino acid changes I95I, Y144T, Y145S and the insertion 146 N in the N-terminal domain, R346K, E484K and N501Y in the Receptor binding Domain (RBD) and P681H in the S1/S2 cleavage site of the Spike protein. The rapid increase in frequency and fixation in a relatively short time in Magdalena, Atlantico, Bolivar, Bogotá D.C, and Santander that were near the theoretical herd immunity suggests an epidemiologic impact. Further studies will be required to assess the biological and epidemiologic roles of the substitution pattern found in the B.1.621 lineage.

Novel Highly Divergent SARS-CoV-2 Lineage With the Spike Substitutions L249S and E484K.

COVID-19 pandemics has led to genetic diversification of SARS-CoV-2 and the appearance of variants with potential impact in transmissibility and viral escape from acquired immunity. We report a new and highly divergent lineage containing 21 distinctive mutations (10 non-synonymous, eight synonymous, and three substitutions in non-coding regions). The amino acid changes L249S and E484K located at the CTD and RBD of the Spike protein could be of special interest due to their potential biological role in the virus-host relationship. Further studies are required for monitoring the epidemiologic impact of this new lineage.

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.

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.

Overexpression of MAP2 and NF-H Associated with Dendritic Pathology in the Spinal Cord of Mice Infected with Rabies Virus.

Rabies is a viral infection that targets the nervous system, specifically neurons. The clinical manifestations of the disease are dramatic and their outcome fatal; paradoxically, conventional histopathological descriptions reveal only subtle changes in the affected nervous tissue. Some researchers have considered that the pathophysiology of rabies is based more on biochemical changes than on structural alterations, as is the case with some psychiatric diseases. However, we believe that it has been necessary to resort to other methods that allow us to analyze the effect of the infection on neurons. The Golgi technique is the gold standard for studying the morphology of all the components of a neuron and the cytoskeletal proteins are the structural support of dendrites and axons. We have previously shown, in the mouse cerebral cortex and now with this work in spinal cord, that rabies virus generates remarkable alterations in the morphological pattern of the neurons and that this effect is associated with the increase in the expression of two cytoskeletal proteins (MAP2 and NF-H). It is necessary to deepen the investigation of the pathogenesis of rabies in order to find therapeutic alternatives to a disease to which the World Health Organization classifies as a neglected disease.

Naturally-occurring porphyrins in a spontaneous-tumour bearing mouse model.

An increase in naturally-occurring porphyrins has been described in the blood of subjects bearing different kinds of tumours, that has been proposed as an additional parameter for the diagnosis of occult cancer, although at present the reason for the phenomenon is not exactly defined. In this work the increase of porphyrins in plasma of tumour-bearing subjects has been investigated in parallel with their occurrence in other tissues, considering the systemic iron homeostasis subversion taking place in the presence of cancer. The transgenic female MMTV-neu mouse-developing spontaneous mammary adenocarcinoma has been used as an experimental model, in comparison to non-transgenic C1 mouse as a control. The spleen, accomplishing both hemocatheretic and hemopoietic functions in rodents, and the liver have been considered because of their deep engagement in heme metabolism, entailing both the fate of protoporphyrin IX (PpIX) as its ultimate precursor, and iron homeostasis. Investigations have been performed by means of microspectrofluorometric and image analysis of tissue autofluorescence (AF), and histochemical detection of non-heme iron. In tumour-bearing mouse, along with a marked PpIX presence in tumour, a PpIX enhancement in spleen and liver is observed, that is accompanied by a significant increase in plasma. The phenomenon can be related to a systemic alteration of heme metabolism induced by tumour cells to face their survival and proliferation requirements.

[Production and evaluation of an antiserum for immunohistochemical detection of rabies virus in aldehyde fixed brain tissues]. / Elaboración y evaluación de un antisuero para la detección inmunohistoquímica del virus de la rabia en tejido cerebral fijado en aldehídos.

INTRODUCTION: The standard procedure for rabies diagnosis requires fresh samples of infected brain to be analyzed by two techniques, direct immunofluorescence and inoculation in mice. Rabies-infected, aldehyde-fixed brain tissues can be examined by immunohistochemistry, but the required commercial antibodies are scarce and expensive. OBJECTIVES: An anti-rabies antiserum was produced and tested to evaluate the effectiveness of rabies antigen detection in aldehyde preserved brain tissue. MATERIALS AND METHODS: Rabbits were inoculated with a rabies vaccine produced in Vero cells (origin-African green monkey kidney). Anti-rabies antiserum was obtained and tested by immunohistochemistry in aldehyde-fixed brain sections of rabies-infected mice. Several experimental conditions were assayed. The usefulness of the antiserum in human pathology samples was also tested. RESULTS: The specificity of the antiserum was demonstrated for immunohistochemical detection of rabies antigen in fixed aldehydes nervous tissue both from experimental material and pathology archival collection. In addition, the antiserum was successful in detecting rabies virus under conditions that have been considered unfavorable for the preservation of antigens. CONCLUSIONS: The inoculation of rabies vaccine in rabbits is an easy and safe procedure for obtaining antiserum useful for the detection of rabies antigen in samples of nervous tissue. Sections obtained on vibratome better preserve the viral antigenicity in comparison with paraffin-embedded tissues. This methods permit less expensive and more rapid immunohistochemical diagnosis of rabies.

Elaboración y evaluación de un antisuero para la detección inmunohistoquímica del virus de la rabia en tejido cerebral fijado en aldehídos / Production and evaluation of an antiserum for immunohistochemical detection of rabies virus in aldehyde fixed brain tissues

Introducción. El procedimiento estándar para el diagnóstico de la rabia requiere de muestras frescas del cerebro infectado, que se estudian mediante las técnicas de inmunofluorescencia directa y la inoculación en ratones. No obstante, a veces se necesita estudiar cerebros infectados con rabia mediante inmunohistoquímica de material fijado en aldehídos, pero los anticuerpos comerciales que se requieren son escasos y costosos. Objetivos. Elaborar un antisuero antirrábico y probar su efectividad para reconocer antígenos de rabia en tejido cerebral preservado en aldehídos. Materiales y métodos. Se inocularon conejos con una vacuna antirrábica producida en células Vero. Se obtuvo un antisuero que fue probado mediante inmunohistoquímica en cortes de cerebro de ratones infectados con rabia, obtenidos en diferentes condiciones experimentales y fijados en aldehídos. Además, se ensayó su utilidad en material de colección histopatológica de casos de rabia humana. Resultados. Se demostró la especificidad del antisuero obtenido para la detección inmunohistoquímica de antígenos de la rabia en tejido nervioso fijado con aldehídos y procedente de material experimental y del archivo histopatológico. Además, el antisuero resultó ser útil para la detección del virus rábico en condiciones que se consideran desfavorables para la preservación de antígenos. Conclusiones. La inoculación de vacuna antirrábica en conejos es un procedimiento fácil y seguro para la obtención de antisuero útil para la detección de antígenos de la rabia en muestras de tejido nervioso. Los cortes obtenidos con vibrátomo preservan mejor la antigenicidad en comparación con los tejidos incluidos en parafina, y permiten acortar el tiempo para hacer el diagnóstico inmunohistoquímico de la rabia.

Introduction. The standard procedure for rabies diagnosis requires fresh samples of infected brain to be analyzed by two techniques, direct immunofluorescence and inoculation in mice. Rabies-infected, aldehyde-fixed brain tissues can be examined by immunohistochemistry, but the required commercial antibodies are scarce and expensive. Objectives. An anti-rabies antiserum was produced and tested to evaluate the effectiveness of rabies antigen detection in aldehyde preserved brain tissue. Materials and methods. Rabbits were inoculated with a rabies vaccine produced in Vero cells (origin-African green monkey kidney). Anti-rabies antiserum was obtained and tested by immunohistochemistry in aldehyde-fixed brain sections of rabies-infected mice. Several experimental conditions were assayed. The usefulness of the antiserum in human pathology samples was also tested. Results. The specificity of the antiserum was demonstrated for immunohistochemical detection of rabies antigen in fixed aldehydes nervous tissue both from experimental material and pathology archival collection. In addition, the antiserum was successful in detecting rabies virus under conditions that have been considered unfavorable for the preservation of antigens. Conclusions. The inoculation of rabies vaccine in rabbits is an easy and safe procedure for obtaining antiserum useful for the detection of rabies antigen in samples of nervous tissue. Sections obtained on vibratome better preserve the viral antigenicity in comparison with paraffin-embedded tissues. This methods permit less expensive and more rapid immunohistochemical diagnosis of rabies.