VP1 is the primary determinant of neuropathogenesis in a mouse model of enterovirus D68 acute flaccid myelitis.

Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory and neurologic disease [acute flaccid myelitis (AFM)]. Intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with US/IL/14-18952 (IL52), a clinical isolate from the 2014 EV-D68 epidemic, results in many of the pathogenic features of human AFM, including viral infection of the spinal cord, death of motor neurons, and resultant progressive paralysis. In distinction, CA/14-4231 (CA4231), another clinical isolate from the 2014 EV-D68 outbreak, does not cause paralysis in mice, does not grow in the spinal cord, and does not cause motor neuron loss following IM injection. A panel of chimeric viruses containing sequences from IL52 and CA4231 was used to demonstrate that VP1 is the main determinant of EV-D68 neurovirulence following IM injection of neonatal SW mice. VP1 contains four amino acid differences between IL52 and CA4231. Mutations resulting in substituting these four amino acids (CA4231 residues into the IL52 polyprotein) completely abolished neurovirulence. Conversely, mutations resulting in substituting VP1 IL52 amino acid residues into the CA4231 polyprotein created a virus that induced paralysis to the same degree as IL52. Neurovirulence following infection of neonatal SW mice with parental and chimeric viruses was associated with viral growth in the spinal cord. IMPORTANCE: Emerging viruses allow us to investigate mutations leading to increased disease severity. Enterovirus D68 (EV-D68), once the cause of rare cases of respiratory illness, recently acquired the ability to cause severe respiratory and neurologic disease. Chimeric viruses were used to demonstrate that viral structural protein VP1 determines growth in the spinal cord, motor neuron loss, and paralysis following intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with EV-D68. These results have relevance for predicting the clinical outcome of future EV-D68 epidemics as well as targeting retrograde transport as a potential strategy for treating virus-induced neurologic disease.

Mycobacterium abscessus Meningitis Associated with Stem Cell Treatment During Medical Tourism.

Mycobacterium abscessus infections have been reported as adverse events related to medical tourism. We report M. abscessus meningitis in a patient who traveled from Colorado, USA, to Mexico to receive intrathecal stem cell injections as treatment for multiple sclerosis. We also review the management of this challenging central nervous system infection.

Efficacy of an isoxazole-3-carboxamide analog of pleconaril in mouse models of Enterovirus-D68 and Coxsackie B5.

Enteroviruses (EV) cause a number of life-threatening infectious diseases. EV-D68 is known to cause respiratory illness in children that can lead to acute flaccid myelitis. Coxsackievirus B5 (CVB5) is commonly associated with hand-foot-mouth disease. There is no antiviral treatment available for either. We have developed an isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of EV-D68 (IC50 58 nM) as well as other enteroviruses including the pleconaril-resistant Coxsackievirus B3-Woodruff (IC50 6-20 nM) and CVB5 (EC50 1 nM). Cryo-electron microscopy structures of EV-D68 in complex with 11526092 and pleconaril demonstrate destabilization of the EV-D68 MO strain VP1 loop, and a strain-dependent effect. A mouse respiratory model of EV-D68 infection, showed 3-log decreased viremia, favorable cytokine response, as well as statistically significant 1-log reduction in lung titer reduction at day 5 after treatment with 11526092. An acute flaccid myelitis neurological infection model did not show efficacy. 11526092 was tested in a mouse model of CVB5 infection and showed a 4-log TCID50 reduction in the pancreas. In summary, 11526092 represents a potent in vitro inhibitor of EV with in vivo efficacy in EV-D68 and CVB5 animal models suggesting it is worthy of further evaluation as a potential broad-spectrum antiviral therapeutic against EV.

Telaprevir Treatment Reduces Paralysis in a Mouse Model of Enterovirus D68 Acute Flaccid Myelitis.

In 2014, 2016, and 2018, the United States experienced unprecedented spikes in pediatric cases of acute flaccid myelitis (AFM), which is a poliomyelitis-like paralytic illness. Accumulating clinical, immunological, and epidemiological evidence has identified enterovirus D68 (EV-D68) as a major causative agent of these biennial AFM outbreaks. There are currently no available FDA-approved antivirals that are effective against EV-D68, and the treatment for EV-D68-associated AFM is primarily supportive. Telaprevir is an food and drug administration (FDA)-approved protease inhibitor that irreversibly binds the EV-D68 2A protease and inhibits EV-D68 replication in vitro. Here, we utilize a murine model of EV-D68 associated AFM to show that early telaprevir treatment improves paralysis outcomes in Swiss Webster (SW) mice. Telaprevir reduces both viral titer and apoptotic activity in both muscles and spinal cords at early disease time points, which results in improved AFM outcomes in infected mice. Following intramuscular inoculation in mice, EV-D68 infection results in a stereotypic pattern of weakness that is reflected by the loss of the innervating motor neuron population, in sequential order, of the ipsilateral (injected) hindlimb, the contralateral hindlimb, and then the forelimbs. Telaprevir treatment preserved motor neuron populations and reduced weakness in limbs beyond the injected hindlimb. The effects of telaprevir were not seen when the treatment was delayed, and toxicity limited doses beyond 35 mg/kg. These studies are a proof of principle, provide the first evidence of benefit of an FDA-approved antiviral drug with which to treat AFM, and emphasize both the need to develop better tolerated therapies that remain efficacious when administered after viral infections and the development of clinical symptoms. IMPORTANCE Recent outbreaks of EV-D68 in 2014, 2016, and 2018 have resulted in over 600 cases of a paralytic illness that is known as AFM. AFM is a predominantly pediatric disease with no FDA-approved treatment, and many patients show minimal recovery from limb weakness. Telaprevir is an FDA-approved antiviral that has been shown to inhibit EV-D68 in vitro. Here, we demonstrate that a telaprevir treatment that is given concurrently with an EV-D68 infection improves AFM outcomes in mice by reducing apoptosis and viral titers at early time points. Telaprevir also protected motor neurons and improved paralysis outcomes in limbs beyond the site of viral inoculation. This study improves understanding of EV-D68 pathogenesis in the mouse model of AFM. This study serves as a proof of principle for the first FDA-approved drug that has been shown to improve AFM outcomes and have in vivo efficacy against EV-D68 as well as underlines the importance of the continued development of EV-D68 antivirals.

Treatment with Granulocyte-Macrophage Colony-Stimulating Factor Reduces Viral Titers in the Brains of West Nile Virus-Infected Mice and Improves Survival.

West Nile virus (WNV) is the leading cause of epidemic arboviral encephalitis in the United States. As there are currently no proven antiviral therapies or licensed human vaccines, understanding the neuropathogenesis of WNV is critical for rational therapeutic design. In WNV-infected mice, the depletion of microglia leads to enhanced viral replication, increased central nervous system (CNS) tissue injury, and increased mortality, suggesting that microglia play a critical role in protection against WNV neuroinvasive disease. To determine if augmenting microglial activation would provide a potential therapeutic strategy, we administered granulocyte-macrophage colony-stimulating factor (GM-CSF) to WNV-infected mice. Recombinant human GM-CSF (rHuGMCSF) (sargramostim [Leukine]) is an FDA-approved drug used to increase white blood cells following leukopenia-inducing chemotherapy or bone marrow transplantation. Daily treatment of both uninfected and WNV-infected mice with subcutaneous injections of GM-CSF resulted in microglial proliferation and activation as indicated by the enhanced expression of the microglia activation marker ionized calcium binding adaptor molecule 1 (Iba1) and several microglia-associated inflammatory cytokines, including CCL2 (C-C motif chemokine ligand 2), interleukin 6 (IL-6), and IL-10. In addition, more microglia adopted an activated morphology as demonstrated by increased sizes and more pronounced processes. GM-CSF-induced microglial activation in WNV-infected mice was associated with reduced viral titers and apoptotic activity (caspase 3) in the brains of WNV-infected mice and significantly increased survival. WNV-infected ex vivo brain slice cultures (BSCs) treated with GM-CSF also showed reduced viral titers and caspase 3 apoptotic cell death, indicating that GM-CSF specifically targets the CNS and that its actions are not dependent on peripheral immune activity. Our studies suggest that stimulation of microglial activation may be a viable therapeutic approach for the treatment of WNV neuroinvasive disease. IMPORTANCE Although rare, WNV encephalitis poses a devastating health concern, with few treatment options and frequent long-term neurological sequelae. Currently, there are no human vaccines or specific antivirals against WNV infections, so further research into potential new therapeutic agents is critical. This study presents a novel treatment option for WNV infections using GM-CSF and lays the foundation for further studies into the use of GM-CSF as a treatment for WNV encephalitis as well as a potential treatment for other viral infections.

Monkeypox-Associated Central Nervous System Disease: A Case Series and Review.

OBJECTIVE: Monkeypox virus (MPXV) disease has been declared a public health emergency by the World Health Organization, creating an urgent need for neurologists to be able to recognize, diagnosis, and treat MPXV-associated neurologic disease. METHODS: Three cases of MPXV-associated central nervous system (CNS) disease occurring during the 2022 outbreak, and their associated imaging findings are presented, with 2 cases previously published in a limited capacity in a public health bulletin. RESULTS: Three previously healthy immunocompetent gay men in their 30s developed a febrile illness followed by progressive neurologic symptoms with presence of a vesiculopustular rash. MPXV nucleic acid was detected by polymerase chain reaction (PCR) from skin lesions of 2 patients, with the third patient having indeterminate testing but an epidemiologic link to a confirmed MPXV disease case. Cerebrospinal fluid demonstrated a lymphocytic pleocytosis, elevated protein, and negative MPXV-specific PCR. In 2 patients, magnetic resonance imaging of the brain and spine demonstrated partially enhancing, longitudinally extensive central spinal cord lesions with multifocal subcortical, basal ganglia, thalamic, cerebellar, and/or brainstem lesions. The third patient had thalamic and basal ganglia lesions. All patients received 14 days of tecovirimat, and 2 patients also received multiple forms of immunotherapy, including intravenous immunoglobulin, pulsed high-dose steroids, plasmapheresis, and/or rituximab. Good neurologic recovery was observed in all cases. INTERPRETATION: MPXV can be associated with CNS disease. It is unclear whether this is from a parainfectious immune-mediated injury or direct CNS viral invasion. ANN NEUROL 2023;93:893-905.

Herpesvirus-Associated Encephalitis: an Update.

Purpose of Review: Herpesviruses are a leading cause of encephalitis worldwide. The article reviews the eight human herpesviruses with a focus on recent advances as they pertain to encephalitis. Recent Findings: Notable recent updates include the development of multiplex polymerase chain reaction (PCR)-based panels, which have improved access to PCR tests, especially in rural and resource-limited areas. Despite unchanged treatment recommendations, research is ongoing into novel therapies. There have been recent advances in vaccines, particularly for varicella zoster virus (VZV) which may impact neurologic complications. Finally, the recent discovery of an association between herpes encephalitis and post-infectious autoimmune encephalitis has had a critical impact on the fields of infectious and autoimmune neurology, though there remains much to learn. Summary: Most herpesviruses are neurotropic and must be considered on the differential diagnosis for infectious encephalitis. This article describes recent advances in the diagnosis, treatment, complications, and management of these infections.

Two Cases of Monkeypox-Associated Encephalomyelitis - Colorado and the District of Columbia, July-August 2022.

Monkeypox virus (MPXV) is an orthopoxvirus in the Poxviridae family. The current multinational monkeypox outbreak has now spread to 96 countries that have not historically reported monkeypox, with most cases occurring among gay, bisexual, and other men who have sex with men (1,2). The first monkeypox case in the United States associated with this outbreak was identified in May 2022 in Massachusetts (1); monkeypox has now been reported in all 50 states, the District of Columbia (DC), and one U.S. territory. MPXV is transmitted by close contact with infected persons or animals; infection results in a febrile illness followed by a diffuse vesiculopustular rash and lymphadenopathy. However, illness in the MPXV current Clade II outbreak has differed: the febrile prodrome is frequently absent or mild, and the rash often involves genital, anal, or oral regions (3,4). Although neuroinvasive disease has been previously reported with MPXV infection (5,6), it appears to be rare. This report describes two cases of encephalomyelitis in patients with monkeypox disease that occurred during the current U.S. outbreak. Although neurologic complications of acute MPXV infections are rare, suspected cases should be reported to state, tribal, local, or territorial health departments to improve understanding of the range of clinical manifestations of and treatment options for MPXV infections during the current outbreak.

Neurology of Acute Viral Infections.

As specialists in acute neurology, neurohospitalists are often called upon to diagnose and manage acute viral infections affecting the nervous system. In this broad review covering the neurology of several acute viral infections, our aim is to provide key diagnostic and therapeutic pearls of practical use to the busy neurohospitalist. We will review acute presentations, diagnosis, and treatment of human herpesviruses, arboviruses, enteroviruses, and some vaccine-preventable viruses. The neurological effects of coronaviruses, including COVID-19, are not covered in this review.

Nervous System Manifestations of Arboviral Infections.

Purpose of Review: Complex environmental factors and human intervention influence the spread of arthropod vectors and the cycle of transmission of arboviruses. The spectrum of clinical manifestations is diverse, ranging from serious presentations like viral hemorrhagic fever (e.g., dengue, yellow fever, rift valley fever) or shock syndromes (e.g., dengue virus) to organ-specific illness like meningoencephalitis. Recent Findings: A spectrum of clinical neurologic syndromes with potential acute devastating consequences or long-term sequelae may result from some arboviral infections. Summary: In this review, we describe some of the most frequent and emerging neuro-invasive arboviral infections, spectrum of neurologic disorders including encephalitis, meningitis, myelitis or poliomyelitis, acute demyelinating encephalomyelitis, Guillain-Barré syndrome, and ocular syndromes.

An Overview of Monkeypox Virus and Its Neuroinvasive Potential.

Monkeypox virus (MPV) is an orthopox virus in the Poxviridae family that is currently of international concern. It is endemic to Central and Western Africa with two known viral clades. Various African rodents and primates are likely the natural reservoirs. Zoonotic transmission occurs by direct contact with infected animals (e.g., bites, scratches, slaughtering). Human to human transmission occurs through close contact with infected persons (e.g., respiratory droplets, skin-on-skin, or sexual contact) or fomites. Classically, human MPV disease first has a febrile prodrome with lymphadenopathy followed by a diffuse maculopapular to vesiculopustular skin/mucosal lesion eruption. In the current 2022 outbreak, which is primarily affecting men who have sex with men (MSM) currently, the febrile prodrome may be absent and skin/mucosal lesions may be isolated to the genital and anal regions. Rarely, MPV likely has the potential to be neuroinvasive based on animal models, previous case series, and preliminary reports currently under investigation. Even though neurologic manifestations of human MPV infection are rare, given the sheer numbers of increasing cases throughout the world, neurologists should be prepared to recognize, diagnose, and treat potential neuroinvasive disease or other neurologic symptoms. ANN NEUROL 2022;92:527-531.

Neutralizing Antibody Given after Paralysis Onset Reduces the Severity of Paralysis Compared to Nonspecific Antibody-Treated Controls in a Mouse Model of EV-D68-Associated Acute Flaccid Myelitis.

Enterovirus D68 (EV-D68) can cause mild to severe respiratory illness and is associated with a poliomyelitis-like paralytic syndrome called acute flaccid myelitis (AFM). Most cases of EV-D68-associated AFM occur in young children who are brought to the clinic after the onset of neurologic symptoms. There are currently no known antiviral therapies for AFM, and it is unknown whether antiviral treatments will be effective if initiated after the onset of neurologic symptoms (when patients are likely to present for medical care). We developed a "clinical treatment model" for AFM, in which individual EV-D68-infected mice are tracked and treated with an EV-D68-specific human-mouse chimeric monoclonal antibody after the onset of moderate paralysis. Mice treated with antibody had significantly better paralysis outcomes compared to nonspecific antibody-treated controls. Treatment did not reverse paralysis that was present at the time of treatment initiation but did slow the further loss of function, including progression of weakness to other limbs, as well as reducing viral titer in the muscle and spinal cords of treated animals. We observed the greatest therapeutic effect in EV-D68 isolates which were neutralized by low concentrations of antibody, and diminishing therapeutic effect in EV-D68 isolates which required higher doses of antibody for neutralization. This work supports the use of virus-specific immunotherapy for the treatment of AFM. It also suggests that patients who present with AFM should be treated as soon as possible if recent infection with EV-D68 is suspected.

Depletion of Microglia in an Ex Vivo Brain Slice Culture Model of West Nile Virus Infection Leads to Increased Viral Titers and Cell Death.

West Nile virus (WNV) is a major cause of viral encephalitis in the United States. WNV infection of the brain leads to neuroinflammation characterized by activation of microglia, the resident phagocytic cells of the central nervous system (CNS). In this study, depletion of CNS microglia using the CSF1R antagonist PLX5622 increased the viral load in the brain and decreased the survival of mice infected with WNV (strain TX02). PLX5622 was also used in ex vivo brain slice cultures (BSCs) to investigate the role of intrinsic neuroinflammatory responses during WNV infection. PLX5622 effectively depleted microglia (>90% depletion) from BSCs resulting in increased viral titers (3 to 4-fold increase in PLX5622-treated samples) and enhanced virus-induced caspase 3 activity and cell death. Microglia depletion did not result in widespread alterations in cytokine and chemokine production in either uninfected or WNV infected BSCs. The results of this study demonstrated how microglia contribute to limiting viral growth and preventing cell death in WNV infected BSCs but were not required for the cytokine/chemokine response to WNV infection. This study highlighted the importance of microglia in the protection from neuroinvasive WNV infection and demonstrated that microglia responses were independent of WNV-induced peripheral immune responses. IMPORTANCE WNV infections of the CNS are rare but can have devastating long-term effects. There are currently no vaccines or specific antiviral treatments, so a better understanding of the pathogenesis and immune response to this virus is crucial. Previous studies have shown microglia to be important for protection from WNV, but more work is needed to fully comprehend the impact these cells have on neuroinvasive WNV infections. This study used PLX5622 to eliminate microglia in an ex vivo brain slice culture (BSC) model to investigate the role of microglia during a WNV infection. The use of BSCs provided a system in which immune responses innate to the CNS could be studied without interference from peripheral immunity. This study will allow for a better understanding of the complex nature of microglia during viral infections and will likely impact the development of new therapeutics that target microglia.