Single-cell RNA-seq landscape midbrain cell responses to red spotted grouper nervous necrosis virus infection.

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS.

Replication of early and recent Zika virus isolates throughout mouse brain development.

Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus-associated injury. Therefore, a critical aspect of ZIKV-induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and midgestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly elongated basal processes of the radial glial progenitor cells and impairment of postmitotic neuronal migration, were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.

Neuroanatomical circuitry between kidney and rostral elements of brain: a virally mediated transsynaptic tracing study in mice.

The identity of higher-order neurons and circuits playing an associative role to control renal function is not well understood. We identified specific neural populations of rostral elements of brain regions that project multisynaptically to the kidneys in 3-6 days after injecting a retrograde tracer pseudorabies virus (PRV)-614 into kidney of 13 adult male C57BL/6J strain mice. PRV-614 infected neurons were detected in a number of mesencephalic (e.g. central amygdala nucleus), telencephalic regions and motor cortex. These divisions included the preoptic area (POA), dorsomedial hypothalamus (DMH), lateral hypothalamus, arcuate nucleus (Arc), suprachiasmatic nucleus (SCN), periventricular hypothalamus (PeH), and rostral and caudal subdivision of the paraventricular nucleus of the hypothalamus (PVN). PRV-614/Tyrosine hydroxylase (TH) double-labeled cells were found within DMH, Arc, SCN, PeH, PVN, the anterodorsal and medial POA. A subset of neurons in PVN that participated in regulating sympathetic outflow to kidney was catecholaminergic or serotonergic. PRV-614 infected neurons within the PVN also contained arginine vasopressin or oxytocin. These data demonstrate the rostral elements of brain innervate the kidney by the neuroanatomical circuitry.

Evidence for developmental dopaminergic alterations in the human immunodeficiency virus-1 transgenic rat.

Neurologic impairments associated with human immunodeficiency virus (HIV) infection in pediatric patients may affect quality of life, and can develop despite antiretroviral therapy (ART). Behavioral changes observed in clinical studies of HIV-infected children suggest alterations in dopaminergic neurotransmission. Findings from our model of choice, the HIV-1 transgenic rat, reveal a significant increase in phosphorylated tyrosine hydroxylase protein expression and a decrease in dopamine transporter mRNA, without changes in tyrosine hydroxylase (TH) or dopamine transporter (DAT) protein or in more general markers of protein and gene expression levels in the HIV-1 transgenic rat midbrain. Thus these findings suggest selective vulnerability of the dopamine system in developing brains to HIV-1 infection.

Midbrain and spinal cord magnetic resonance imaging (MRI) changes in poliomyelitis.

Poliomyelitis, though eradicated from most parts of the world, continues to occur in India. There is paucity of data on the magnetic resonance imaging (MRI) changes in poliomyelitis. We report a 3(1/2)-year-old boy who presented with subacute onset flaccid paralysis and altered sensorium. Stool culture was positive for wild polio virus type 3. Magnetic resonance imaging revealed signal changes in bilateral substantia nigra and anterior horns of the spinal cord. These MRI changes may be of potential diagnostic significance in a child with poliomyelitis.

Some observations on the tropism of Japanese encephalitis virus in rat brain.

The clinical picture of viral encephalitis is determined by the affinity and persistence of the virus to different brain regions. Therefore, the present study was aimed to investigate the neuropathological changes following Japanese encephalitis virus (JEV) infection in rat at different time points. Twelve days old Wistar rats were infected by intracerebral inoculation of JEV. Presence of JEV antigen was detected in thalamus, striatum, cortex and mid brain on 3, 6, 10 and 20 days post inoculation (d.p.i.). Histopathological changes were also studied in different brain regions at different time points. The highest expression of JEV antigen was found on 6 dpi in all the brain regions studied. JEV antigen was maximum in thalamus on 6 d.p.i. and mid brain on 10 d.p.i. JEV antigen, however, was almost undetectable on 20 d.p.i. in all the regions. The classical pathological changes such as cellular infiltration, perivascular cuffing, meningeal disruption, neuronal damage, neuronal shrinkage, and plaque formation were observed up to 10 d.p.i. The present study reveals high affinity of JEV to thalamus, brainstem and striatum. Rat model of JEV infection may serve as a useful model for studying mechanism of cell injury and recovery in JE.

Preferential sensitivity of human dopaminergic neurons to gp120-induced oxidative damage.

The dopamine (DA)-rich midbrain is known to be a key target of human immunodeficiency virus (HIV)-1. Studies of simian immunodeficiency virus (SIV)-induced neuropathogenesis recently established that there is a major disruption within the nigrostriatal dopaminergic system characterized by marked depletion of dopaminergic neurons, microglial cell activation, and reactive astrocytes. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, experiments were performed to characterize the damage to dopaminergic neurons induced by HIV-1 gp120. Functional impairment was assessed by DA uptake, and neurotoxicity was measured by apoptosis and oxidative damage. Through the use of this mesencephalic neuronal/glial culture model, we were able to identify the relative sensitivity of dopaminergic neurons to gp120-induced damage, manifested as reduced function (decreased DA uptake), morphological changes, and reduced viability. We also showed that gp120-induced oxidative damage is involved in this neuropathogenic process.

Neural circuits controlling diaphragm function in the cat revealed by transneuronal tracing.

Although a number of studies have considered the neural circuitry that regulates diaphragm activity, these pathways have not been adequately discerned, particularly in animals such as cats that utilize the respiratory muscles during a variety of different behaviors and movements. The present study employed the retrograde transneuronal transport of rabies virus to identify the extended neural pathways that control diaphragm function in felines. In all animals deemed to have successful rabies virus injections into the diaphragm, large, presumed motoneurons were infected in the C(4)-C(6) spinal segments. In addition, smaller presumed interneurons were labeled bilaterally throughout the cervical and upper thoracic spinal cord. While in short and intermediate survival cases, infected interneurons were concentrated in the vicinity of phrenic motoneurons, in late survival cases, the distribution of labeling was more expansive. Within the brain stem, the earliest infected neurons included those located in the classically defined pontine and medullary respiratory groups, the medial and lateral medullary reticular formation, the region immediately ventral to the spinal trigeminal nucleus, raphe pallidus and obscurus, and the vestibular nuclei. At longer survival times, infection appeared in the midbrain, which was concentrated in the lateral portion of the periaqueductal gray, the region of the tegmentum that contains the locomotion center, and the red nucleus. Considerable labeling was also present in the fastigial nucleus of the cerebellum, portions of the posterior and lateral hypothalamus and the adjacent fields of Forel known to contain hypocretin-containing neurons and the precruciate gyrus of cerebral cortex. These data raise the possibility that several parallel pathways participate in regulating the activity of the feline diaphragm, which underscores the multifunctional nature of the respiratory muscles in this species.

Presence of corticotrophin-releasing factor and/or tyrosine hydroxylase in cells of a neural brain-testicular pathway that are labelled by a transganglionic tracer.

Our laboratory has shown that male testosterone levels are not solely controlled by the release of hypothalamic gonadotrophin-releasing hormone and pituitary luteinising hormone, but are also regulated by a multisynaptic pathway connecting the brain and the testis that interferes with the testosterone response to gonadotrophins. This pathway, which is independent of the pituitary gland, is activated by an i.c.v. injection of either the stress-related peptide corticotrophin-releasing factor (CRF) or of beta-adrenoceptor agonists, both of which alter androgen release and decrease levels of the peripheral-type benzodiazepine receptor and the steroidogenic acute regulatory protein within Leydig cells. Our original studies used the retrograde transganglionic tracer pseudorabies virus (PRV) to map progression of the virus from the testes to upper brain levels. The present study aimed to extend this work by identifying the regions where CRF and catecholamine neurones represented components of the stress-activated, brain-testicular pathway that prevents testosterone increases. To this end, anaesthetised adult male rats received an intra-testicular injection of PRV. Using immunofluorescence, we identified co-labelling of PRV and either CRF or tyrosine hydroxylase (TH), the enzyme responsible for biogenic amine synthesis. Co-labelling of PRV and CRF was found in the bed nucleus of the stria terminalis, the paraventricular nucleus of the hypothalamus (PVN) and the central amygdala. Co-labelling of PRV and TH was found in the PVN, substantia nigra, A7/Kölliker-Fuse area, area of A5, locus coeruleus, nucleus of solitary tract, area of C3, area of C2 and the area of C1/A1. These results indicate that most cell groups of the ventral noradrenergic pathway have neurones that are a part of the brain-testicular pathway. This suggests that the stress hormones CRF and catecholamines may act as neurotransmitters that signal the pathway to inhibit increases in plasma testosterone levels.

Effect of exogenous interferon and an interferon inducer on western equine encephalitis virus disease in a hamster model.

Mice are used as models for western equine encephalitis virus (WEEV) infection, but high mortality is generally only seen with intracranial or intranasal challenge, while peripheral inoculation results in approximately 50% mortality and is not dose-dependent. Hamsters were therefore studied as a model for WEEV infection. Hamsters were highly sensitive to intraperitoneal (i.p.) infection with WEEV. Disease progression was rapid, and virus titers in serum, brain, liver, and kidney of infected hamsters peaked between 2 and 4 days post-virus inoculation (dpi). Foci of virus infection were detected in neurons of the cerebral cortex and midbrain. Pre-treatment i.p. with either interferon alfacon-1 (5 microg/kg/day) or with Ampligen (3.2 mg/kg/day) resulted in complete survival, reduced brain titers, and improved weight gain. This model of WEEV infection in hamsters appears to serve as a suitable model for the evaluation of potential therapeutic agents for the treatment of WEE disease.

West Nile virus encephalitis causing fatal CNS toxicity after hematopoietic stem cell transplantation.

We describe here a patient who died of progressive CNS deterioration following allogeneic stem cell transplant with West Nile virus as the sole pathogen on the cerebrospinal fluid and brain tissue analysis. A 50-year-old male with Philadelphia chromosome-positive acute lymphocytic leukemia (ALL) underwent allogeneic PBSCT from his HLA identical sister. After engraftment, the patient developed fever with progressive and ultimately fatal neurological deterioration. Imaging studies of the brain including CT and MRI scans were remarkable for mild low attenuation lesions of the white matter. CSF analysis was negative for neoplastic cells, bacteria, AFB, CMV, HSV, fungal infections and leukemic relapse. However, serological analysis of both the serum and CSF was positive for West Nile virus-specific IgM antibodies. At autopsy, West Nile virus PCR and cultures were positive in the mid-brain tissue. Electron micrographs showed evidence of viral particles. Given the recent increase in the spread of West Nile virus infections and the increased susceptibility of BMT patients to infectious complications, West Nile virus encephalitis should be considered in patients undergoing transplantation.

Platelet-derived growth factor-producing cells immortalized from rat mesencephalon with SV40 large T antigen transduced by an AAV vector.

PURPOSE: Adeno-associated virus (AAV) can infect a wide variety of mammalian cell types and is capable of infecting both dividing and non-dividing cell populations. Here we report the construction of a recombinant AAV vector which expresses the SV40 large T protein (AAV-T) and the use of this vector to immortalize primary cells from embryonic rat mesencephalon. METHODS: The AAV-T vector was constructed by introducing the BamH1 fragment of the pCMV/SVE/Neo plasmid containing T antigen and SV40 regulatory elements into the JM48 plasmid containing the inverted terminal repeats of AAV. Neuronal cultures from E-12 rat mesencephalon were grown in defined media supplemented with basic fibroblast growth factor. These cells were infected with the AAV-T vector. RESULTS: A cell line (designated RMAT) and six subclones were established from these cultures through multiple passages. This cell line was immunoreactive for SV40 large T antigen and the cytoskeletal proteins nestin and vimentin. Morphological differentiation and expression of neurofilament 160 kDa were induced by exposure to dibutyrl cyclic AMP. Immunoassays performed to measure endogenous production of growth factors showed that RMAT cells produced high levels of platelet-derived growth factor (PDGF). CONCLUSIONS: AAV may be a useful vector for the transduction of oncogenes to produce cell lines.

Persistence of the influenza A/WSN/33 virus RNA at midbrain levels of immunodefective mice.

Strains of influenza A virus are known to infect specific subpopulations of neurons in the mouse brain. Here we report that all segments of the genome of the neurotropic influenza A virus, strain WSN/33, can persist in the brains of immunodefective transporter associated with Antigen Processing 1 (TAP1) mutant mice. Ten to 17 months after injection of virus into the olfactory bulbs, viral RNA encoding the nonstructural NS1 protein was detected in sections from the brain at midbrain levels by RT-PCR in almost all animals. Both negative-strand genomic RNA (vRNA) and positive-strand RNA, including mRNA, were found. RNA encoding nucleoprotein and polymerases, which form the replicative complex of the virus, were detected in fewer brains. RNA encoding envelope proteins were found only in occasional brains. No viral cDNA could be identified. This observation shows that certain regions of the brain in immunodefective mice may harbor the genome of influenza A virus including the NS1 gene, the products of which may play a regulatory role in host-cell metabolism.

Demonstration of Akabane virus antigen using immunohistochemistry in naturally infected newborn calves.

Eight newborn calves showing ataxia were necropsied and examined histologically. Six of seven cerebrospinal fluid samples collected from these animals had neutralizing antibody for Akabane virus (AKV). All examined calves had nonsuppurative encephalomyelitis, localized mainly in the midbrain and spinal cord. Corresponding to the encephalitic lesion, AKV antigen was demonstrated in neuroglial cells in the brain stem and neuronal cells in the ventral horn of the spinal cord. This is the first study to demonstrate AKV antigen by immunohistochemistry in naturally infected newborn calves.

Markedly severe dystonia in Japanese encephalitis.

Encephalitis has been reported to be a rare cause of severe dystonia. We describe five patients with markedly severe dystonia from Japanese encephalitis. These patients with markedly severe dystonia were seen during the past 8 years as a subgroup of 50 patients with Japanese encephalitis. The diagnosis of markedly severe dystonia was based on increasingly frequent episodes of generalized dystonia with bulbar, respiratory, or metabolic derangement or leading to exhaustion or pain. The diagnosis of JE was based on clinicoradiologic features and a fourfold increase of hemagglutination-inhibiting antibody titers in paired serum. The outcome of the patients was defined as a good, partial, or poor recovery on the basis of 1-year clinical status. All the patients were males, and their ages ranged from 6 to 19 years. Movement disorders appeared 1 to 3 weeks after the illness as the level of consciousness started improving. During the next 1 to 4 weeks, patients began to experience markedly severe dystonia. It was associated with marked axial dystonia resulting in opisthotonus and retrocollis in five patients, jaw-opening dystonia in two patients, teeth clenching in one patient, and oculogyric crisis and neck deviation in another patient. The attacks of markedly severe dystonia lasted for 2 to 30 minutes and occurred as many as 20 to 30 times daily. Other developments included fixed limb dystonia in one patient, severe spasticity and rigidity in five patients, and focal muscle wasting in one patient. These patients had only a modest improvement after treatment. Markedly severe dystonia abated by 2 to 6 months in all the patients who were followed up. Cranial magnetic resonance imaging showed bilateral thalamic involvement in all patients, brainstem involvement in three patients, and basal ganglia involvement in two patients. At the 3-month follow-up, all patients had a poor outcome. At 1 year, one patient had a complete recovery; one had a partial recovery; and two were bedridden. It can be concluded that markedly severe dystonia is an important and serious sequela of Japanese encephalitis and may occur as the result of thalamus, midbrain, or basal ganglia involvement in various combinations.

A hamster model of equine herpesvirus 9 induced encephalitis.

An acute and lethal infection of equine herpesvirus 9 (EHV-9), a new type of equine herpesvirus, was established in Syrian hamsters by intranasal inoculation. Clinical symptoms included the loss of body weight, nasal and ocular discharges and apparent neurological symptoms. Both LD50 and ID50 were equal at 33 plaque forming units. Histological and immunohistochemical examination demonstrated that the virus replicated in the olfactory mucosal cells and in the neurons of the olfactory bulbs, cerebrum and mesencephalon. The induction of encephalitis by intranasal but not by other routes of inoculation (i.v., i.p., i.m.) indicated that EHV-9 entered the brain via the olfactory nerve and then spread trans-synaptically to connecting neurons along the olfactory tract. This animal model should be useful for studying the pathogenesis and neurovirulence of this newly discovered neurotropic virus as well as other neurotropic herpesviruses.

Neuronal labeling in the rat brain and spinal cord from the ovary using viral transneuronal tracing technique.

In the present investigations the viral transneuronal labeling method, which is able to reveal hierarchial chains of central nervous system (CNS) neurons, was applied to identify sites in the CNS connected with the ovary and presumably involved in the control of ovarian functions. Pseudorabies virus was injected into the ovaries of rats and a few days later (at various times after the injection) the spinal cord and brain were examined for virus-infected neurons from the ovary. The virus-labeled nerve cells were identified by immunocytochemistry using polyclonal antiviral antibody. Virus-labeled neurons were detected both in the spinal cord and the brain. In the spinal cord such elements were observed in the intermediolateral cell column, in the dorsal horn close to the marginal zone and in the central autonomic nucleus. In the medulla oblongata and pons, neurons of several nuclei and cell groups (area postrema, nucleus of the solitary tract, dorsal vagal complex, nucleus ambiguus, paragigantocellular nucleus, parapyramidal nucleus, A1, A5 and A7 cell groups, caudal raphe nuclei, locus ceruleus, subceruleus nucleus, Barrington's nucleus, Kölliker-Fuse nucleus) were found to be transneuronally labeled. In the mesencephalon, the ventrolateral part of the periaqueductal gray matter contained virus-labeled neurons. In the diencephalon, a very intensive cell body labeling was observed in the hypothalamic paraventricular nucleus and a few virus-infected neurons could be detected in the lateral and dorsal hypothalamus, in the arcuate nucleus, zona incerta, perifornical area and in the anterior hypothalamus. Concerning the telencephalic structures, virus-labeled cells were found in the bed nucleus of the stria terminalis and in the central amygdala nucleus. These findings provide the first neuromorphological evidence for the existence of a multisynaptic neuronal pathway between the ovary and the CNS, and give a detailed account of the structures involved in this pathway.

Adenoviral-mediated transfection of the Lac-Z gene into rat dissociated embryonic central nervous system cells before and after seeding.

The adenovirus carrying a reporter gene--the Lac Z gene--is known to infect central nervous system (CNS) cells in primary cell cultures. The percentage of infected neurons with respect to the total number of neurons was studied in primary dissociated cultures as a function of the day of inoculation and the age of three rat CNS cultures: spinal cord, mesencephalon and cortex. Two methods of viral inoculation were compared: the first inoculation was performed on the cultured cell at 2, 3 or 6 days in vitro (DIV) whereas the second inoculation was performed on the cell suspensions before seeding. All the infected CNS cells has the same aspect as the control cultures. In the spinal cord and the mesencephalic cultures, the glial cells were preferentially infected, especially when the cells were inoculated at 6 DIV. In the cortical cultures, there were more infected neurons than infected glial cells. The number of CNS cells was lower when inoculation was performed at 6 DIV as compared with 3 DIV. Very few infected GABA cells were found in the cultures. A high percentage of infected neuronal cells relative to the total number of neuronal cells was found when infection of the three types of cultures was performed on the dissociated embryonic cell suspension before seeding.