Studies on the pathogenesis of and propagation of enterovirus 71 in poliomyelitis-like disease in monkeys

Summary Poliomyelitis-like disease in monkeys infected with enterovirus 71 (E71) were studied to determine whether nerve cell damage is due primarily to virus multiplication in the neurons or secondary to the effects of virus multiplication in the supporting tissue of the central nervous system (CNS). Monkeys infected with E71 develop a disease with neuromuscular or muscular dysfunction and lesions of the CNS. E71 was recovered from the lumbar and cervical cord and from the cerebrum. Specific immunofluorescence was detected in the degenerating or necrotic nerve cells in the anterior grey horns of the lumbar and cervical cord, the cerebellum and cerebrum, although virus antigen was not found in the white matter. No specific immunofluorescence was found in the glial cells, vascular endothelial or mononuclear inflammatory cells. From the present results, it is suggested that nerve cell damage is due principally to virus multiplication in the neurons of the CNS in monkeys infected with E71.     

Productive simian virus 40 infection of neurons in immunosuppressed Rhesus monkeys

There currently is no animal model of JC virus-associated progressive multifocal leukoencephalopathy (PML). Reactivation of simian virus 40 (SV40) in immunosuppressed rhesus monkeys, however, rarely causes a PML-like illness. We sought to isolate a neurotropic clone of SV40 and determine its pathogenic potential in monkeys. The clone SV40CNS1 was amplified by polymerase chain reaction from the brain DNA of a simian/human immunodeficiency virus-infected monkey that had developed PML and meningoencephalitis. Compared with the SV40 prototype 776, SV40CNS1 had a small number of single-amino-acid mutations and caused a productive infection in monkey fibroblasts. It was inoculated into 2 SV40-negative, simian/human immunodeficiency virus-immunosuppressed monkeys. Both animals developed meningoencephalitis with productive SV40 infection of cerebral cortical neurons and glia in the superficial layers of the cortex and at the gray-white junction. Focal SV40-infected cells were also found in the cerebellar molecular and granule cell layers and white matter. Both animals also developed disseminated SV40 infection with nephritis and pneumonitis. Thus, SV40CNS1 is infectious and pathogenic in immunosuppressed monkeys, but it induces encephalitis with fulminant productive infection in cortical neurons and systemic disease, rather than PML. These findings shed new light on SV40 neurotropism and expand the host cell range of this virus.     

Pathologic characterization of a murine model of human enterovirus 71 encephalomyelitis

We describe a model of Enterovirus 71 encephalomyelitis in 2-week-old mice that shares many features with the human central nervous system (CNS) disease. Mice were infected via oral and parenteral routes with a murine-adapted virus strain originally from a fatal human case. The mice succumbed to infection after 2 to 5 days. Vacuolated and normal-appearing CNS neurons showed viral RNA and antigens and virions by in situ hybridization, immunohistochemistry, and electron microscopy; inflammation was minimal. The most numerous infected neurons were in anterior horns, motor trigeminal nuclei, and brainstem reticular formation; fewer neurons in the red nucleus, lateral cerebellar nucleus, other cranial nerve nuclei, motor cortex, hypothalamus, and thalamus were infected. Other CNS regions, dorsal root, and autonomic ganglia were spared. Intramuscular-inoculated mice killed 24 to 36 hours postinfection had viral RNA and antigens in ipsilateral lumbar anterior horn cells and adjacent axons. Upper cord motor neurons, brainstem, and contralateral motor cortex neurons were infected from 48-72 hours. Viral RNA and antigens were abundant in skeletal muscle and adjacent tissues but not in other organs. The distinct, stereotypic viral distribution in this model suggests that the virus enters the CNS via peripheral motor nerves after skeletal muscle infection, and spread within the CNS involves motor and other neural pathways. This model may be useful for further studies on pathogenesis and for testing therapies.     

Comparative studies on the neurovirulence of temperature-sensitive and temperature-resistant viruses of enterovirus 71 in monkeys

The neurovirulence of strain BrCr of enterovirus 71 (E71) was compared in monkeys between temperature-sensitive (ts) and temperature-resistant (tr) viruses. Comparisons are made relative to clinical disease, pathologic findings, serum neutralizing antibody titers, CNS virus replication as measured by infectivity titrations and immunofluorescence. Clinically, ts virus did not produce a clinical disease. The tr virus, however, produced paralysis. Pathologically, little or no nerve cell damage was found in the CNS of monkeys inoculated with ts virus, although mild to moderate interstitial changes occurred. In monkeys inoculated with tr virus, marked nerve cell damage and inflammatory reaction were found in the CNS. Serum neutralizing antibody titers in monkeys inoculated with ts virus rose on day 25. No virus was detected in the CNS of monkeys inoculated with ts virus, while a high virus titer was detected in the CNS of monkeys inoculated with tr virus. No specific immunofluorescence was detected in the nerve cells of the CNS in monkeys inoculated with ts virus, but specific fluorescence was detected in the nerve cells of the CNS in monkeys inoculated with tr virus. Virus growth in the CNS correlated well with the severity of clinical and pathologic findings, and immunofluorescent studies. The results show that ts virus was much less neurovirulent than tr virus, indicating that ts virus resembles the attenuated poliovirus which could not grow at a higher temperature. It is inferred that the genetic factors which influence the reproductive capacity of E71 at a higher temperature are very closely correlated with the neurovirulence.     

Ultrastructural studies on the pathogenesis of poliomyelitis in monkeys infected with polivirus

Summary Poliovirus was inoculated intraspinally into cynomolgus monkeys to determine whether nerve cell damage in the central nervous system (CNS) is due primarily to virus multiplication in the neuron or to secondary effects of virus multiplication in the supporting cells. Electron-microscopically, the development of cytopathogenesis and of membrane-bound vesicles and virus particles in the neurons of the CNS in monkeys infected with poliovirus was compared with that of infected cultured cynomolgus monkey kidney (CMK) cells. The structure of membrane-bound vesicles in cytoplasm of damaged motoneuron was examined and found to be similar to the vesicles in infected cultured CMK cells. Virus-like particles were detected occasionally around or within membrane-bound vesicles in the cytoplasm of degenerating motoneurons as well as cultured CMK cells, although intracytoplasmic crystals were not detected in the neuron. No virus particles or membrane-bound vesicles were found in astrocyte foot plates, microglia, oligodendrocytes, axons, vascular endothelial, and inflammatory cells. In addition, poliovirus antigen was detected only in the nerve cells of the CNS by the immunoperoxidase technique, although specific staining was never found in the supporting tissues. From the present results we suggest that membrane-bound vesicles in the cytoplasm of the motoneuron are closely correlated with virus multiplication and that damage of the nerve cell is due to the direct action of the poliovirus.     

Toxic effects of MDMA on central serotonergic neurons in the primate: importance of route and frequency of drug administration

This study compared the toxic effects of oral versus subcutaneous and single versus multiple doses of 3,4-methylenedioxymethamphetamine (MDMA) on central serotonergic neurons in non-human primates. Orally administered MDMA was approximately one-half as effective as subcutaneously administered drug. Multiple doses were more effective than single doses, but a single 5 mg/kg dose of MDMA given orally still produced a long-lasting depletion of serotonin in the monkey brain. These results indicate that when MDMA is given to monkeys in a manner similar to that employed by humans, it exerts toxic effects on central serotonergic neurons. This suggests that humans using MDMA may be at risk for incurring central serotonergic neuronal damage.     

Remyelination of central nervous system lesions in experimental genital herpes simplex virus infection

To study spinal cord remyelination in a model of genital herpes simplex virus type 2 (HSV-2) infection, adult female mice were inoculated by a vaginal route. At intervals up to 6 months after infection, cord tissues were removed and examined by light and electron microscopy and by immunohistochemical methods. As a consequence of acute infection, 60% of mice developed multifocal central nervous system (CNS) demyelinative lesions in the lower thoracic, lumbar, or upper sacral cord. These lesions, already present 10 days after infection, contained naked axons and mononuclear cells, including macrophages. At 2 weeks, while active myelin breakdown was still ongoing, numerous Schwann cells were present in lesions and surrounded denuded axons. At 3 weeks, the earliest remyelination was seen, and was carried out by Schwann cells and to a lesser extent by oligodendrocytes. Remyelination was extensive by 6-10 weeks and was apparently completed after 3 months. Immunocytochemical studies using antisera to myelin proteins showed relatively distinct zones of central and peripheral remyelination in some lesions, whereas remyelination was of mixed type in others. Thus the remyelinative response following experimental HSV-2-induced CNS demyelination begins promptly, proceeds briskly and goes to completion. With a natural route of inoculation and a relatively avirulent strain of this human pathogen, we have produced a model of CNS white matter injury and repair in a high proportion of infected mice that may be useful in understanding mechanisms of human demyelinative disease.     

Differential development and electrophysiological activity in cultured cortical neurons from the mouse and cynomolgus monkey

In vitro cultures of primary cortical neurons are widely used to investigate neuronal function. However, it has yet to be fully investigated whether there are significant differences in development and function between cultured rodent and primate cortical neurons, and whether these differences influence the utilization of cultured cortical neurons to model pathological conditions. Using in vitro culture techniques combined with immunofluorescence and electrophysiological methods, our study found that the development and maturation of primary cerebral cortical neurons from cynomolgus monkeys were slower than those from mice. We used a microelectrode array technique to compare the electrophysiological differences in cortical neurons, and found that primary cortical neurons from the mouse brain began to show electrical activity earlier than those from the cynomolgus monkey. Although cultured monkey cortical neurons developed slowly in vitro, they exhibited typical pathological features-revealed by immunofluorescent staining-when infected with adeno-associated viral vectors expressing mutant huntingtin(HTT), the Huntington's disease protein. A quantitative analysis of the cultured monkey cortical neurons also confirmed that mutant HTT significantly reduced the length of neurites. Therefore, compared with the primary cortical neurons of mice, cultured monkey cortical neurons have longer developmental and survival times and greater sustained physiological activity, such as electrophysiological activity. Our findings also suggest that primary cynomolgus monkey neurons cultured in vitro can simulate a cell model of human neurodegenerative disease, and may be useful for investigating time-dependent neuronal death as well as treatment via neuronal regeneration. All mouse experiments and protocols were approved by the Animal Care and Use Committee of Jinan University of China(IACUC Approval No. 20200512-04) on May 12, 2020. All monkey experiments were approved by the IACUC protocol(IACUC Approval No. LDACU 20190820-01) on August 23, 2019 for animal management and use.     

Neural mechanisms for navigation

To examine the neural basis of navigation we recorded the activity of neurons in the monkey medial parietal region (MPR) while monkeys actively navigated through a virtual environment. We found navigation neurons whose responses to the same movement at the same location were modulated depending on the route that the monkey was currently taking, that is, in a route-selective manner. The reversible inactivation of MPR neurons by muscimol resulted in a monkey becoming lost during the navigation task trial. These results suggest that MPR plays a critical role in navigation by integrating location information and self-movement information.     

Resistance of rat CNS to brain stem infection with herpes simplex virus type 1

Infection of the CNS by herpes simplex type 1 (HSV-1) via the trigeminal route to the brain stem was elucidated in a rat model. In contrast to the earlier described cortical and hippocampal infection after intracranial injection, the CNS showed a profound resistance to HSV-1 infection when the virus was administred by nose inoculation, as judged by histopathology and immunohistochemistry. In contrast, when the distribution of HSV-1 in the brain was investigated after nose inoculation by polymerase chain reaction, viral DNA was detected at all levels from the ganglia to the cortex. When replication of HSV-1 was assayed in primary cell cultures of rat astrocytes derived from brain stem, striatum, hippocampus and cerebral cortex, significantly lower virus yields were obtained in brain stem-derived astrocytes cultures as compared with in cortex-derived astrocytes. This finding was independent of whether HSV-1 strains used originated from brains of patients suffering from herpes simplex encephalitis or from patients with oral cutaneous lesions and lacking neurological symptoms. Also, by immunocytochemistry of cultures after HSV-1 infection, a lower number of plaques were seen in brain stem-derived astrocytes as compared with cortex-derived astrocytes. The observed relative resistance of brain stem-derived astrocytes to replicate HSV-1 might contribute to the ability of the brain stem to withstand infection during reactivation of this virus in the trigeminal neurons.Financial support was received from the Swedish Medical Research Council, the Medical Society of Göteborg, the Swedish Medical Society, and the Faculty of Medicine, University of Göteborg     

Experimental rabies virus infection in Artibeus jamaicensis bats with CVS-24 variants

An experimental model of rabies was established in the fruit-eating bat species Artibeus jamaicensis. The infections caused by CVS-N2c and CVS-B2c, which are both stable variants of CVS-24, were compared after inoculation of adult bats in the right masseter muscle. CVS-N2c produced neurologic signs of rabies with paresis, ataxia, and inability to fly, while CVS-B2c did not produce neurologic signs. Bats were sacrificed and the distribution of rabies virus antigen was assessed in tissue sections with immunoperoxidase staining. Both viruses spread to the brain stem and bilaterally to the trigeminal ganglia by days 2 to 3. CVS-N2c had disseminated widely in the central nervous system (CNS) by day 4 and had involved the spinal cord, thalamus, cerebellum, and cerebral cortex. CVS-B2c had infected neurons in the spinal cord on day 5 and in the cerebellum, thalamus, and cerebral cortex on day 6. Infected pyramidal neurons of the hippocampus were observed on day 5 in CVS-N2c infection, but infected neurons were never noted in the hippocampus in CVS-B2c infection. CVS-N2c infected many more neurons and more prominently involved neuronal processes than CVS-B2c. CVS-N2c spread more efficiently in the CNS than CVS-B2c. Morphologic changes of apoptosis or biochemical evidence of DNA fragmentation were not observed in neurons with either virus after this route of inoculation. The different neurovirulent properties of these CVS variants in this model were not related to their in vivo ability to induce apoptosis.     

Restricted posterior parietal lesions in the rhesus monkey and performance on visuospatial tasks

Monkeys with lesions restricted to the inferior parietal lobule or the banks and depths of the superior temporal sulcus were tested on a route-following task. These areas are considered on neuroanatomical grounds to be homologous to parts of the human posterior parietal cortex, where lesions produce profound spatial disorientation. The operated monkeys were impaired on the route task, thus confirming at the behavioural level the anatomical predictions of comparability between parietal cortex in monkey and that in man. The monkeys were not impaired, however, on a visual pattern discrimination or on a visual-spatial task with cue and response separation, a task that was considered on the basis of previous investigations with extensive posterior lesions in the monkey to be sensitive to parietal lesions.     

Plasma gelsolin accumulates in macrophage nodules in brains of simian immunodeficiency virus infected rhesus macaques

Plasma gelsolin (pGSN), an isoform 1, is secreted by various types of cells in the central nervous system (CNS) and periphery, but not by the liver. pGSN circulates in blood and cerebrospinal fluid (CSF); however, its concentration in CSF is approximately twenty times lower than in plasma. It has been shown that several types of cells such as oligodendrocytes, neurons, and/or astrocytes contribute to the overall pool of pGSN in the CNS. Further, it has been postulated that pGSN plays multiple roles during microbial infection and modulates inflammatory responses; however, the exact mechanism of regulation is not known. We previously showed that levels of pGSN in CSF of individuals with advanced neurocognitive impairment due to HIV infection of the brain are decreased. Here, we show that macrophages express significant amounts of pGSN in response to HIV infection in vitro. Using immunohistochemistry of simian immunodeficiency virus infected rhesus monkey brains, we show that increased levels of pGSN are present in macrophage nodules creating locally a high level of this protein within the brain. This may not be reflected by the overall decreased level in the distinct CSF compartment.     

Epileptic and normal neurons in monkey neocortex: A quantitative study of degree of operant control

The object of this study was to quantify and compare the degree of control monkeys may assert over firing patterns of normal and epileptic neurons. Thirty-seven epileptic and 70 normal neurons were studied in detail. The operant task was for the monkey to generate specified consecutive interspike intervals (ISI). The monkeys demonstrated far greater accuracy in controlling consecutive ISIs of normal neurons and were only able to control the intervals between bursts from epileptic neurons. The data implies that high frequency bursts of action potentials from epileptic neurons are all-or-nothing events initiated by synaptic mechanisms. In addition, some data are from a monkey with epilepsia partialis continua; in comparison to less active foci, this focus was comprised of a higher percentage of 'pacemaker' epileptic neurons.     

Changes in cathepsins B-1 and D, neutral proteinase and 2',3'-cyclic nucleotide-3'-phosphohydrolase activities in monkey brain with experimental allergic encephalomyelitis

Myelin basic protein modified with 2′-hydroxy-5′-nitrobenzylbromide or homologous monkey white matter was used to induce experimental allergic encephalomyelitis (EAE) in rhesus monkeys. Altogether 7 monkeys with and without EAE were studied. Macroscopic lesions (plaques), normal-appearing white matter of normal rhesus monkey brains were removed and assayed for cathepsins B-1 and D, neutral proteinase and 2′-3′-cyclic nucleotide-3′-phosphohydrolase activities. Lymph nodes from 2 monkeys, 1 with and 1 without EAE were also included in the present investigation.Cathepsins B-1 and D and neutral proteinase were significantly increased in lesion areas of EAE monkeys when compared with the findings in normal brain material. The enzyme 2′,3′-cyclic nucleotide-3′-phosphohydrolase was found to be significantly decreased in the lesions when compared to the normal-appearing white matter areas of the same animals. The change parallels the reduction of other well-established constituents of the myelin, giving support to the concept that the enzyme is a component of the myelin.The results of the enzyme assays are discussed in relation to the pathogenesis of CNS lesions in EAE.     

Effect of cyclosporin A on an experimental chronic viral infection of the central nervous system

The effects of cyclosporin A (CsA) on neuropathological lesions induced by a chronic viral infection have been tested in the experimental model of the mouse hepatitis virus 3 (MHV3) infection. Daily injections of CsA (50 mg/kg) inhibited the expression of the MHV3-induced ependymitis, meningitis, hydrocephalus and vasculitis. The effect was preserved even if CsA treatment was initiated 15 days after virus infection but was lost if CsA treatment was given later on or for a shorter period of time. Viral titers in brains of chronically infected mice were not affected by CsA treatment. During the first week following MHV3 infection, CsA treatment increased both the percentage of acute death (31 vs. 10%) and the viral titers in brain and liver of infected mice. In this model, the timing of CsA treatment appeared critical for the balance between its beneficial effect on CNS lesions and the risk of increased acute mortality.     

Protective action of recombinant neurturin on dopaminergic neurons in substantia nigra in a rhesus monkey model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease characterized by muscular trembling palsy due to lack of dopamine (DA) in the substantia nigra-striatum (nigrostriatal) system resulting from the degeneration and necrosis of dopaminergic neurons. No effective cure has been found. Neurturin (NTN) has been demonstrated to act specifically on midbrain (mesencephalic) dopaminergic neurons with protective actions specifically. In the present study, we induced rhesus monkey model of Parkinson's disease by injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Rhesus monkeys were randomly divided into a PD model group, NTN treatment group and normal control groups. In the NTN treatment group, 1 mg of E. coli-derived recombinant human NTN was injected into the cerebral ventricles 48 h before the injection of MPTP. Results indicated that Rhesus monkeys in the PD model group acquired PD symptoms that increasingly aggravated over time, while monkeys treated with NTN had less apparent or no symptoms. Using fluorospectrophotometry, the dopamine (DA), 5, 5-hydroxytrytamine (5-HT) and the 5-hydroxyindoleacetic acid (5-HIAA) contents of DA, 5-HT and 5-HIAA in substantia nigra, putamen and caudate nucleus in monkeys from the model group was found to be significantly lower than in the normal control group. While no significant differences were found between monkeys treated with NTN and normal control groups, the contents of DA, 5-HT and 5-HIAA in the NTN treatment group were higher than those observed in the PD model group. A dramatic loss of neurons in the substantia nigra in monkeys in the PD model group was observed by light microscopy, while no obvious loss was observed in the NTN treatment group in which the numbers of neurons were similar to those in normal controls. These results indicate that recombinant human NTN can prevent PD symptoms as well as protect dopaminergic neurons and preserve DA content in midbrain substantia nigra in rhesus monkeys exposed to MPTP.