Outbreaks Of Acute Flaccid Myelitis Research Articles

Enterovirus D-68 Infection of Primary Rat Cortical Neurons: Entry, Replication, and Functional Consequences.

Enterovirus D68 (EV-D68) is an emerging pathogen associated with mild to severe respiratory disease. Since 2014, EV-D68 is also linked to acute flaccid myelitis (AFM), causing paralysis and muscle weakness in children. However, it remains unclear whether this is due to an increased pathogenicity of contemporary EV-D68 clades or increased awareness and detection of this virus. Here, we describe an infection model of primary rat cortical neurons to study the entry, replication, and functional consequences of different EV-D68 strains, including historical and contemporary strains. We demonstrate that sialic acids are important (co)receptors for infection of both neurons and respiratory epithelial cells. Using a collection of glycoengineered isogenic HEK293 cell lines, we show that sialic acids on either N-glycans or glycosphingolipids can be used for infection. Additionally, we show that both excitatory glutamatergic and inhibitory GABA-ergic neurons are susceptible and permissive to historical and contemporary EV-D68 strains. EV-D68 infection of neurons leads to the reorganization of the Golgi-endomembranes forming replication organelles, first in the soma and later in the processes. Finally, we demonstrate that the spontaneous neuronal activity of EV-D68-infected neuronal network cultured on microelectrode arrays (MEA) is decreased, independent of the virus strain. Collectively, our findings provide novel insights into neurotropism and -pathology of different EV-D68 strains, and argue that it is unlikely that increased neurotropism is a recently acquired phenotype of a specific genetic lineage. IMPORTANCE Acute flaccid myelitis (AFM) is a serious neurological illness characterized by muscle weakness and paralysis in children. Since 2014, outbreaks of AFM have emerged worldwide, and they appear to be caused by nonpolio enteroviruses, particularly enterovirus-D68 (EV-D68), an unusual enterovirus that is known to mainly cause respiratory disease. It is unknown whether these outbreaks reflect a change of EV-D68 pathogenicity or are due to increased detection and awareness of this virus in recent years. To gain more insight herein, it is crucial to define how historical and circulating EV-D68 strains infect and replicate in neurons and how they affect their physiology. This study compares the entry and replication in neurons and the functional consequences on the neural network upon infection with an old "historical" strain and contemporary "circulating" strains of EV-D68.

Infections of the Spine and Spinal Cord.

Infections of the spine and spinal cord are associated with a high risk of morbidity and mortality and, therefore, require prompt clinical recognition, efficient diagnostic evaluation, and interdisciplinary treatment. This article reviews the pathophysiology, epidemiology, clinical manifestations, diagnosis, and treatment of infections of the spine and spinal cord to help practicing clinicians recognize, evaluate, and manage patients with such infections. Aging of the population, increasing use of immunosuppressive medications, and other factors have contributed to increasing rates of spinal infections. Although the most common agents responsible for spinal infections remain bacteria and viruses, fungal infections occur in individuals who are immunocompromised, and parasitic infections are common in endemic regions, but patterns are in evolution with migration and climate change. Recent outbreaks of acute flaccid myelitis in children have been associated with enteroviruses A71 and D68. Infections of the spine and spinal cord can be challenging to diagnose, requiring a thorough history and neurologic examination, laboratory studies of serum and CSF, neuroimaging (particularly MRI), and, in some instances, biopsy, to establish a diagnosis and treatment regimen. Interdisciplinary management including collaboration with experts in internal medicine, infectious disease, and neurosurgery is important to improve clinical outcomes.

Respiratory and intestinal epithelial cells exhibit differential susceptibility and innate immune responses to contemporary EV-D68 isolates.

Enterovirus D68 (EV-D68) has been implicated in outbreaks of severe respiratory illness and is associated with acute flaccid myelitis (AFM). EV-D68 is often detected in patient respiratory samples but has also been detected in stool and wastewater, suggesting the potential for both respiratory and enteric routes of transmission. Here, we used a panel of EV-D68 isolates, including a historical pre-2014 isolate and multiple contemporary isolates from AFM outbreak years, to define the dynamics of viral replication and the host response to infection in primary human airway cells and stem cell-derived enteroids. We show that some recent EV-D68 isolates have decreased sensitivity to acid and temperature compared with earlier isolates and that the respiratory, but not intestinal, epithelium induces a robust type III interferon response that restricts infection. Our findings define the differential responses of the respiratory and intestinal epithelium to contemporary EV-D68 isolates and suggest that a subset of isolates have the potential to target both the human airway and gastrointestinal tracts.

Acute flaccid myelitis: long-term outcomes recorded in the CAPTURE study compared with paediatric transverse myelitis

BackgroundSince 2014, the USA has documented three outbreaks of acute flaccid myelitis (AFM). Unique features and treatment responses of this myelitis variant have not been prospectively studied. This study prospectively...

#91: Human Antibodies Neutralize Enterovirus D68 and Protect Against Infection and Paralytic Disease

Abstract Background Enterovirus D68 (EV-D68) causes worldwide outbreaks of human respiratory illness with spatiotemporally related outbreaks of acute flaccid myelitis (AFM). The major circulating clade of EV-D68 has changed since the largest ever recorded outbreak, which occurred in the United States in 2014. Numerous seroepidemiology studies show that essentially all adult humans have EV-D68-neutralizing antibodies in their serum, even in serum collected prior to large outbreaks. But some studies show a lack of neutralizing antibodies in pediatric populations. We sought to isolate human monoclonal antibodies (mAbs) from B cells in peripheral blood mononuclear cells (PBMCs) of immune subjects, induced by natural infection, to further understand human humoral immunity to EV-D68. Methods We obtained PBMCs from donors with known infection during 2014. We used EV-D68 virus isolates from this outbreak as antigen in an indirect ELISA to screen immortalized PBMCs for antigen-specificity, then fused B cells with myeloma cells to create hybridomas. Results We isolated >60 naturally occurring anti-EV-D68 human mAbs. These mAbs exhibit diverse binding affinities when compared across clades of recent EV-D68 isolates. Many mAbs neutralize EV-D68 quite potently in vitro, with [ng/mL] half-maximal effective concentrations. Some mAbs like EV68-228 neutralize diverse clades, whereas others are highly clade-specific. Binding of antibodies to at least three, but likely more, major antigenic sites on the virus leads to neutralization. When given as either prophylaxis before or treatment after EV-D68 inoculation, mAb EV68-228 protects mice from both respiratory and neurologic disease, whereas equivalent doses of polyclonal human immunoglobulin protect only partially. Conclusions We observed a qualitative difference among antibodies isolated from patients who had a history of natural infection. These differences could contribute to certain individuals being susceptible to respiratory disease and AFM. These studies of humoral immunity are especially important for a disease with nearly universal apparent seroprotection, in which the virus somehow persistently causes outbreaks across the world. Furthermore, no licensed vaccines or treatments exist for EV-D68. These mAbs have therapeutic benefit in vivo and show promise for the prevention and/or treatment of EV-D68-related diseases in humans. We are pursuing the use of mAb EV68-228 in humans with partners in industry and government agencies.

Acute flaccid myelitis outbreak through 2016-2018: A multicenter experience from Turkey.

We aim to describe the demographic characteristics, etiology, neurophysiology, imaging findings, treatment, prognosis, and prognostic factors of acute flaccid myelitis. The clinical data, laboratory test and, magnetic resonance imaging (MRI) results of pediatric patients diagnosed with acute flaccid myelitis according to the Centers for Disease Control criteria between August 1, 2016, and December 31, 2018, from 13 centers in Turkey were reviewed. Of the 34 cases identified, 31 were confirmed (91.2%). Eighteen patients (55.9%) were boys. The median patient age was 4 years (interquartile range 2.5-6.9 years). Most of the patients were admitted in 2018 (n=27). A preceding history of a febrile illness was reported in all patients, with a median of 4 days (interquartile range 3-7 days) before symptom onset. Thirty-one patients had T2 hyperintensity on spinal MRI, and 18 patients had cerebrospinal fluid pleocytosis. The most common infectious agents were entero/rhinoviruses (n=5) in respiratory specimens. All patients except one received immunotherapy either alone or in combination. Among 27 patients with follow-up data 24 had persistent weakness. Involvement of four limbs together with an abnormal brain MRI at onset were associated with a poor prognosis. The number of patients with acute flaccid myelitis increased since 2012, spiking with every 2-year interval, largely in the pediatric population. The median age decreases with every outbreak. Clinicians should be aware of the clinical picture for early collection of specimens and early start of rehabilitation programs. Further studies are needed to better characterize the etiology, pathogenesis, risk factors, and treatment of this rare condition.

Acute infectious myelopathies.

The patient who presents with an acute spinal cord syndrome with weakness/paralysis of the limbs presents a diagnostic. Two important syndromes are acute transverse myelitis (ATM) and acute flaccid paralysis (AFP). Both can be caused by a number of infectious and noninfectious causes. Since 2014 there have been outbreaks of acute flaccid myelitis (a subgroup of AFP) in the United States, with a national surveillance program underway. In addition, there have been increasing reports of ATM from new and emerging pathogens, and opportunistic infections in immunocompromised hosts. Infectious causes of ATM or AFP need to be ruled out first. There may be important clues to an infectious cause from epidemiologic risk factors, immune status, international travel, MRI, and laboratory findings. We summarize key features for the more common pathogens in this review. Advances in laboratory testing have improved the diagnostic yield from cerebrospinal fluid, including real-time polymerase chain reaction, metagenomic next-generation sequencing, and advanced antibody detection techniques. These tests still have limitations and require clinical correlation. We present a syndromic approach to infectious myelopathies, focusing on clinical patterns that help narrow the diagnostic possibilities.

Respiratory illness and acute flaccid myelitis in the Tokai district in 2018.

An outbreak of acute flaccid myelitis was chronologically correlated with an outbreak of severe respiratory illness in Japan in 2015. We hypothesized that increases in children hospitalized with severe respiratory illnesses might also be associated with increase in acute flaccid myelitis in autumn 2018. We explored the temporal correlations between respiratory illness outbreaks and acute flaccid myelitis during autumn season between 2016 and 2018 using questionnaire surveys. One questionnaire explored the monthly numbers of children with acute flaccid myelitis, Guillain-Barré syndrome, and other acute flaccid paralyses. The other questionnaire explored the monthly numbers of children hospitalized with respiratory illnesses associated with wheezing. A correlation between the monthly numbers of children with acute flaccid myelitis and those with respiratory illness was analyzed using the Pearson correlation test. Although the number of patients hospitalized with respiratory illness did not correlate with the number of those admitted with myelitis, increases in children aged 7-12 and 13-19years requiring intensive care unit admission correlated temporally with an outbreak of acute flaccid myelitis. An increase in intensive care unit admissions to treat respiratory disease occurred in association with a cluster of acute flaccid myelitis. An increase in the number of intensive care unit admissions due to respiratory illness may be a clue to expect the occurrence of acute flaccid myelitis.

2593. Human Monoclonal Antibodies Potently Neutralize Enterovirus D68 in both a Clade-Specific and -Independent Manner

BackgroundEnterovirus D68 (EV-D68) causes worldwide outbreaks of human respiratory illness with spacio-temporally related outbreaks of acute flaccid myelitis (AFM), a polio-like illness. Numerous seroepidemiology studies show that nearly all humans older than 2 years have EV-D68-neutralizing antibodies in their serum, even in serum collected prior to large outbreaks. However, little else is known about the human antibody response to this virus. We sought to isolate human monoclonal antibodies (mAbs) from B cells in peripheral blood mononuclear cells (PBMCs) of immune subjects, induced by natural infection, to understand human humoral immunity to EV-D68.MethodsWe obtained PBMCs from donors with known infection during the largest ever recorded outbreak, which occurred in the United States in 2014. We used EV-D68 virus isolates from this outbreak in an indirect ELISA to screen immortalized PBMCs for antigen-specificity, then fused them with myeloma cells to create hybridomas.ResultsTo date, we have isolated > 60 naturally occurring anti-EV-D68 human mAbs from the B cells of subjects with documented infection. These mAbs exhibit diverse binding affinities when compared across different clades of recent EV-D68 isolates. Many mAbs neutralize EV-D68 quite potently in vitro, with [ng/mL] half maximal effective concentrations. Some mAbs neutralize diverse clades of EV-D68, whereas others are highly clade-specific. Binding of antibodies to at least three, but likely more, major antigenic sites on the virus leads to neutralization.ConclusionWe observed a qualitative difference among antibodies isolated from patients who had natural infection. These differences could contribute to certain individuals being susceptible to respiratory disease and AFM. Our studies of humoral immunity are especially important for a disease with nearly universal apparent seroprotection, in which the virus somehow persistently causes outbreaks across the world. Furthermore, no licensed vaccines or treatments exist for EV-D68. Given the ability of human intravenous immune globulin to protect mice from AFM, these mAbs are being tested for therapeutic benefit in vivo, and may have promise in the prevention and/or treatment of EV-D68-related diseases.Disclosures All authors: No reported disclosures.

LB14. Cerebrospinal Fluid Profiling of the Human Host Response Reveals Species-Specific Enterovirus Biosignatures in Acute Flaccid Myelitis Cases

BackgroundSince 2014 there have been global biennial outbreaks of acute flaccid myelitis (AFM), a rare but severe “polio-like” illness of as yet-unknown etiology primarily affecting children. Enteroviruses (EVs),, especially EV-D68 and EV-A71, have been implicated in association with AFM cases, but proving causality has been difficult as EVs are rarely isolated from cerebrospinal fluid. In addition, early identification of EV-associated AFM is challenging given that the diagnosis is reliant on potentially subjective clinical and radiological criteria with no specific biomarkers described to date.MethodsWe leveraged existing and newly generated data from a clinical CSF metagenomic assay for pathogen identification at University of California, San Francisco (UCSF) to interrogate the host response at the transcriptome level by RNA sequencing (RNA-Seq). These transcriptome RNA-Seq data were used to create statistical classification models to discriminate among viral infections that have been linked to AFM, including EV-D68, EV-A71, West Nile virus, and Powassan virus. The dynamic range of CSF cellularity (0 to >106 cells/mL), resulting in varying trancriptome coverage, as well as technical variation across samples required the development and validation of novel normalization techniques. In total, we analyzed ~50 CSF samples split into independent training and test sets.ResultsWe were able to demonstrate a distinct signature of AFM that was able to predict the virus associated with AFM in blinded test samples with >80% accuracy. The key transcriptional features that best discriminated EV-A71 from EV-D68-associated AFM involved protein targeting, viral transcription, viral gene expression, and translation initiation pathways.ConclusionHere we demonstrate a novel approach to diagnosis of AFM that relies on host transcriptional biomarkers from cerebrospinal fluid. In the future, this method might allow earlier diagnosis of AFM to drive appropriate therapies and vaccines and predict patient outcomes, as well as guide research studies on the pathophysiology of EV-associated AFM.Disclosures All authors: No reported disclosures.

Contemporary Circulating Enterovirus D68 Strains Infect and Undergo Retrograde Axonal Transport in Spinal Motor Neurons Independent of Sialic Acid.

Enterovirus D68 (EV-D68) is an emerging virus that has been identified as a cause of recent outbreaks of acute flaccid myelitis (AFM), a poliomyelitis-like spinal cord syndrome that can result in permanent paralysis and disability. In experimental mouse models, EV-D68 spreads to, infects, and kills spinal motor neurons following infection by various routes of inoculation. The topography of virus-induced motor neuron loss correlates with the pattern of paralysis. The mechanism(s) by which EV-D68 spreads to target motor neurons remains unclear. We sought to determine the capacity of EV-D68 to spread by the neuronal route and to determine the role of known EV-D68 receptors, sialic acid and intracellular adhesion molecule 5 (ICAM-5), in neuronal infection. To do this, we utilized a microfluidic chamber culture system in which human induced pluripotent stem cell (iPSC) motor neuron cell bodies and axons can be compartmentalized for independent experimental manipulation. We found that EV-D68 can infect motor neurons via their distal axons and spread by retrograde axonal transport to the neuronal cell bodies. Virus was not released from the axons via anterograde axonal transport after infection of the cell bodies. Prototypic strains of EV-D68 depended on sialic acid for axonal infection and transport, while contemporary circulating strains isolated during the 2014 EV-D68 outbreak did not. The pattern of infection did not correspond with the ICAM-5 distribution and expression in either human tissue, the mouse model, or the iPSC motor neurons.IMPORTANCE Enterovirus D68 (EV-D68) infections are on the rise worldwide. Since 2014, the United States has experienced biennial spikes in EV-D68-associated acute flaccid myelitis (AFM) that have left hundreds of children paralyzed. Much remains to be learned about the pathogenesis of EV-D68 in the central nervous system (CNS). Herein we investigated the mechanisms of EV-D68 CNS invasion through neuronal pathways. A better understanding of EV-D68 infection in experimental models may allow for better prevention and treatment strategies of EV-D68 CNS disease.

Outbreaks of acute flaccid myelitis? What neurologists need to know

After a third apparent spike in cases of acute flaccid myelitis across the USA, the US Centers for Disease Control and Prevention is on alert. Neurologists worldwide should be aware of what might be an emerging disease. Adrian Burton reports.

Unusual increase of cases of myelitis in a pediatric hospital in Argentina.

The association between enterovirus D68 and acute flaccid myelitis was first described in the United States in 2014. Since then, sporadic cases have been reported in Canada and Europe. This study describes a series of cases of acute flaccid myelitis at Hospital de Pediatría "Prof. Dr. Juan P. Garrahan," in Buenos Aires, Argentina, during 2016. Descriptive, retrospective study. All patients with acute flaccid myelitis and lesions in the spinal cord involving the gray matter, as observed in the magnetic resonance imaging (MRI) scan, hospitalized from 04/01/2016 to 07/01/2016, were included in the study. Samples of nasopharyngeal secretions, fecal swabs and cerebrospinal fluid were collected and processed to look for the causative agent. Ten patients were included. The median age was 4 years old (range from 3 months to 5 years old). Eight patients had a self-limiting febrile condition before the onset of neurological symptoms. Neurological findings were flaccid weakness in, at least, one limb, cervical paralysis (n= 2) and facial paralysis (n= 2). All patients had longitudinal lesions in the spinal cord, with gray matter involvement, mainly in the anterior horn. In all cases, a lumbar puncture (spinal tap) was performed. Pleocytosis was observed in 7 patients. In four children, enterovirus D68 was identified in nasopharyngeal secretions, and in one, it was detected in the cerebrospinal fluid. Neurological deficit persisted in all patients at the time of discharge. The first outbreak of acute flaccid myelitis associated to enterovirus D68 is reported in Argentina. Active epidemiological surveillance will help to determine the true incidence, epidemiology and etiology of this disease.

2016 Acute Flaccid Myelitis Outbreak in Texas: Promising Outcomes

2016 Acute Flaccid Myelitis Outbreak in Texas: Promising Outcomes

Acute Flaccid Myelitis Outbreak.

Investigators from the University School of Medicine, Salt Lake City, Utah, report an outbreak of acute flaccid myelitis (AFM) occurring in 2014-2015 in several States and reported to the CDC.

Recognition and Management of Acute Flaccid Myelitis in Children

BackgroundIn 2014-2015, several regions of the United States experienced an outbreak of acute flaccid myelitis in pediatric patients. A common, unique feature was disease localization to the gray matter of the spinal cord. MethodsWe report 11 children, ages 13 months to 14 years (median 9 years), in the Intermountain West who presented with extremity weakness (n = 10) or cranial neuropathy (n = 1) of varying severity without an apparent etiology. ResultsAll children experienced acute paralysis, and 10 had symptoms or signs that localized to the spinal cord. Maximum paralysis occurred within 4 days of onset in all patients. All had spinal gray matter lesions consistent with acute myelitis detected by magnetic resonance imaging; no single infectious cause was identified. Despite therapy with intravenous immunoglobulin, corticosteroids, or plasma exchange, nine of 10 (90%) children had motor deficits at follow-up. ConclusionsRecognition of this disorder enables clinicians to obtain appropriate imaging and laboratory testing, initiate treatment, and provide families with accurate prognostic information. In contrast to other causes of acute flaccid paralysis in childhood, most children with acute flaccid myelitis have residual neurological deficits.

Case Definition Expands, Surveillance Increases, But Mystery Remains about Cause of Acute Flaccid Myelitis Outbreak

Case Definition Expands, Surveillance Increases, But Mystery Remains about Cause of Acute Flaccid Myelitis Outbreak

Re-analysis of metagenomic sequences from acute flaccid myelitis patients reveals alternatives to enterovirus D68 infection

Metagenomic sequence data can be used to detect the presence of infectious viruses and bacteria, but normal microbial flora make this process challenging. We re-analyzed metagenomic RNA sequence data collected during a recent outbreak of acute flaccid myelitis (AFM), caused in some cases by infection with enterovirus D68. We found that among the patients whose symptoms were previously attributed to enterovirus D68, one patient had clear evidence of infection with Haemophilus influenzae, and a second patient had a severe Staphylococcus aureus infection caused by a methicillin-resistant strain. Neither of these bacteria were identified in the original study. These observations may have relevance in cases that present with flaccid paralysis because bacterial infections, co-infections or post-infection immune responses may trigger pathogenic processes that may present as poliomyelitis-like syndromes and may mimic AFM. A separate finding was that large numbers of human sequences were present in each of the publicly released samples, although the original study reported that human sequences had been removed before deposition.

Re-analysis of metagenomic sequences from acute flaccid myelitis patients reveals alternatives to enterovirus D68 infection.

Metagenomic sequence data can be used to detect the presence of infectious viruses and bacteria, but normal microbial flora make this process challenging. We re-analyzed metagenomic RNA sequence data collected during a recent outbreak of acute flaccid myelitis (AFM), caused in some cases by infection with enterovirus D68. We found that among the patients whose symptoms were previously attributed to enterovirus D68, one patient had clear evidence of infection with Haemophilus influenzae, and a second patient had a severe Staphylococcus aureus infection caused by a methicillin-resistant strain. Neither of these bacteria were identified in the original study. These observations may have relevance in cases that present with flaccid paralysis because bacterial infections, co-infections or post-infection immune responses may trigger pathogenic processes that may present as poliomyelitis-like syndromes and may mimic AFM. A separate finding was that large numbers of human sequences were present in each of the publicly released samples, although the original study reported that human sequences had been removed before deposition.

Re-analysis of metagenomic sequences from acute flaccid myelitis patients reveals alternatives to enterovirus D68 infection

Metagenomic sequence data can be used to detect the presence of infectious viruses and bacteria, but normal microbial flora make this process challenging. We re-analyzed metagenomic RNA sequence data collected during a recent outbreak of acute flaccid myelitis (AFM), caused in some cases by infection with enterovirus D68. We found that among the patients whose symptoms were previously attributed to enterovirus D68, one patient had clear evidence of infection with Haemophilus influenzae, and a second patient had a severe Staphylococcus aureus infection caused by a methicillin-resistant strain. Neither of these bacteria were identified in the original study. These observations may have relevance in cases that present with flaccid paralysis because bacterial infections, co-infections or post-infection immune responses may trigger pathogenic processes that may present as poliomyelitis-like syndromes and may mimic AFM. A separate finding was that large numbers of human sequences were present in each of the publicly released samples, although the original study reported that human sequences had been removed before deposition.