Enterovirus RNA Research Articles

Molecular epidemiology of enteroviruses associated with hand, foot, and mouth disease in South India from 2015 to 2017

Hand, foot, and mouth disease (HFMD) is a common childhood infection caused by human enteroviruses and is clinically characterised by fever with vesicular rash on the hands, feet, and mouth. While enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) were the major etiological agents of HFMD in India earlier, the data on recently circulating enteroviruses associated with HFMD are sparse. Here, we describe the molecular epidemiology of enteroviruses associated with HFMD in South India from 2015 to 2017. We used archived enterovirus real-time reverse transcription (RT) PCR-positive vesicle swab and/or throat swab specimens from clinically suspected HFMD cases collected from four secondary-care hospitals in South India between July 2015 and December 2017. PCR amplification and sequencing were done based on the 5’VP1, 3’VP1, VP2, or 5´NCR regions to identify enterovirus types. Genetic diversity among enteroviruses was inferred by phylogenetic analysis. Of the 107 enterovirus RNA real-time RT-PCR-positive HFMD cases, 69 (64%) were typed as CVA6, 16 (15%) were CVA16, and one (1%) was CVA10, whereas in 21 (20%) cases, the virus was not typeable by any of the methods used in the study. The majority of HFMD cases (89, 83%) were in children less than five years old, while 11 (10.3%) were in adults. 5’VP1 yielded the maximum number of enteroviruses genotyped, and phylogenetic analysis showed that the CVA6 strains belonged to subclade D3, while the subclades of CVA16 and CVA10 were B1c and D, respectively. The predominant etiological agent of HFMD in South India during 2015-2017 was CVA6, followed by CVA16 and CVA10.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-022-05561-0.

Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage

Aims/hypothesisEnteroviral infection has been implicated consistently as a key environmental factor correlating with the appearance of autoimmunity and/or the presence of overt type 1 diabetes, in which pancreatic insulin-producing beta cells are destroyed by an autoimmune response. Genetic predisposition through variation in the type 1 diabetes risk gene IFIH1 (interferon induced with helicase C domain 1), which encodes the viral pattern-recognition receptor melanoma differentiation-associated protein 5 (MDA5), supports a potential link between enterovirus infection and type 1 diabetes.MethodsWe used molecular techniques to detect enterovirus RNA in peripheral blood samples (in separated cellular compartments or plasma) from two cohorts comprising 79 children or 72 adults that include individuals with and without type 1 diabetes who had multiple autoantibodies. We also used immunohistochemistry to detect the enteroviral protein VP1 in the pancreatic islets of post-mortem donors (n=43) with type 1 diabetes.ResultsWe observed enhanced detection sensitivity when sampling the cellular compartment compared with the non-cellular compartment of peripheral blood (OR 21.69; 95% CI 3.64, 229.20; p<0.0001). In addition, we show that children with autoimmunity are more likely to test positive for enterovirus RNA than those without autoimmunity (OR 11.60; 95% CI 1.89, 126.90; p=0.0065). Furthermore, we found that individuals carrying the predisposing allele (946Thr) of the common variant in IFIH1 (rs1990760, Thr946Ala) are more likely to test positive for enterovirus in peripheral blood (OR 3.07; 95% CI 1.02, 8.58; p=0.045). In contrast, using immunohistochemistry, there was no correlation between the common variant in IFIH1 and detection of enteroviral VP1 protein in the pancreatic islets of donors with type 1 diabetes.Conclusions/interpretationOur data indicate that, in peripheral blood, antigen-presenting cells are the predominant source of enterovirus infection, and that infection is correlated with disease stage and genetic predisposition, thereby supporting a role for enterovirus infection prior to disease onset.Graphical abstract

Enteroviral Infections in the First Three Months of Life.

Enteroviruses (EVs) are an important source of infection in the paediatric age, with most cases concerning the neonatal age and early infancy. Molecular epidemiology is crucial to understand the circulation of main serotypes in a specific area and period due to their extreme epidemiological variability. The diagnosis of EVs infection currently relies on the detection of EVs RNA in biological samples (usually cerebrospinal fluid and plasma, but also throat swabs and feces) through a polymerase chain reaction assay. Although EVs infections usually have a benign course, they sometimes become life threatening, especially when symptoms develop in the first few days of life. Mortality is primarily associated with myocarditis, acute hepatitis, and multi-organ failure. Neurodevelopmental sequelae have been reported following severe infections with central nervous system involvement. Unfortunately, at present, the treatment of EVs infections is mainly supportive. The use of specific antiviral agents in severe neonatal infections has been reported in single cases or studies including few neonates. Therefore, further studies are needed to confirm the efficacy of these drugs in clinical practice.

Enterovirus Sequence Data Obtained from Primate Samples in Central Africa Suggest a High Prevalence of Enteroviruses.

ABSTRACTEnteroviruses infect humans and animals and can cause disease, and some may be transmitted across species barriers. We tested Central African wildlife and found Enterovirus RNA in primates (17) and rodents (2). Some sequences were very similar, while others were dissimilar to known species, highlighting the underexplored enterovirus diversity in wildlife.

Genetic diversity of enteroviruses in patients with respiratory infection

Enterovirus genetic variability underlies the variety of clinical forms of diseases they cause. The aim of the presented study was to establish the genetic diversity of enteroviruses (EVs) that caused acute respiratory infection (ARI) in 2016–2019. Biological samples were obtained from 203 patients with various forms of ARI, EV detection was carried out by RT-PCR, followed by sequencing of the main capsid protein gene and phylogenetic reconstruction. EV RNA was detected in 34.4 % of samples, most often in children aged 1–6 years (53.1–54.8 %). Coxsackieviruses B were found in patients with respiratory enterovirus infection (EVI) significantly more often than other EVs, the dominant serotypes were Coxsackievirus B4, B5. Despite the significant genetic diversity of EVs identified in patients with ARI (three genetic lines of Coxsackievirus B5, two genotypes of Coxsackievirus B2, one genotype of Coxsackievirus B3, three genovariant Coxsackievirus B4, one genovariant Coxsackievirus B1), there is no evidence of their connection with the formation of the respiratory form of EVI.The high level of genetic variability of EVs requires regular molecular-epidemiological surveillance for the identification of emerging genetic variants and assessment of their epidemic potential.

Characterisation of enterovirus RNA detected in the pancreas and other specimens of live patients with newly diagnosed type 1 diabetes in the DiViD study

Aims/hypothesisThe Diabetes Virus Detection (DiViD) study is the first study to laparoscopically collect pancreatic tissue and purified pancreatic islets together with duodenal mucosa, serum, peripheral blood mononuclear cells (PBMCs) and stools from six live adult patients (age 24–35 years) with newly diagnosed type 1 diabetes. The presence of enterovirus (EV) in the pancreatic islets of these patients has previously been reported.MethodsIn the present study we used reverse transcription quantitative real-time PCR (RT-qPCR) and sequencing to characterise EV genomes present in different tissues to understand the nature of infection in these individuals.ResultsAll six patients were found to be EV-positive by RT-qPCR in at least one of the tested sample types. Four patients were EV-positive in purified islet culture medium, three in PBMCs, one in duodenal biopsy and two in stool, while serum was EV-negative in all individuals. Sequencing the 5′ untranslated region of these EVs suggested that all but one belonged to enterovirus B species. One patient was EV-positive in all these sample types except for serum. Sequence analysis revealed that the virus strain present in the isolated islets of this patient was different from the strain found in other sample types. None of the islet-resident viruses could be isolated using EV-permissive cell lines.Conclusions/interpretationEV RNA can be frequently detected in various tissues of patients with type 1 diabetes. At least in some patients, the EV strain in the pancreatic islets may represent a slowly replicating persisting virus.Graphical abstract

Localization of enteroviral RNA within the pancreas in donors with T1D and T1D-associated autoantibodies

SummaryEnteroviral infections have been associated with autoimmunity and type 1 diabetes (T1D), but reliable methods to ascertain localization of single infected cells in the pancreas were missing. Using a single-molecule-based fluorescent in situ hybridization (smFISH) method, we detected increased virus infection in pancreases from organ donors with T1D and with disease-associated autoantibodies (AAb+).Although virus-positive β cells are found at higher frequency in T1D pancreases, compared to control donors, but are scarce, most virus-positive cells are scattered in the exocrine pancreas. Augmented CD45+ lymphocytes in T1D pancreases show virus positivity or localization in close proximity to virus-positive cells. Many more infected cells were also found in spleens from T1D donors.The overall increased proportion of virus-positive cells in the pancreas of AAb+ and T1D organ donors suggests that enteroviruses are associated with immune cell infiltration, autoimmunity, and β cell destruction in both preclinical and diagnosed T1D.

Clinical features and management strategies of Meningitis in adult patients: A hospital based study from Kuwait

Introduction: High mortality rate of meningitis has been reported in Kuwait, however, limited data is available describing this disease in adult patients. With this background, we conducted a prospective study on patients admitted with meningitis, with an objective to describe the risk factors, clinical presentation, disease course and outcome; focus given on diagnostic problems and consequent management difficulties.&#x0D; Methods: Our team diagnosed, managed and documented hospital records of patients (n=44) admitted with suspected meningitis at a referral hospital during 2010-12. Detailed information was collected regarding clinical presentation, CSF analysis, treatment, hospital course and outcome as per Glasgow Outcome Scale (GOS). Results: Bacterial, viral, and tuberculosis (TB) meningitis were seen in 22.8%, 52.3%, and 15.9% of patients. Clinical features of our cohort were consistent with available literature. Positive identification of organism by culture, gram stain, or antigen test was possible in only 6 patients i.e. Streptococcus pneumonae-(n=3), Streptococcus agalactiae-(n=2), Neisseria meningitides-(n=1). CSF polymerase chain reaction was detected positive for Enterovirus RNA, and Herpes simplex virus DNA for one patient each. Empirical antimicrobial treatment directed against common causative organisms was the mainstay of management of bacterial meningitis. Viral meningitis was managed symptomatically. TB meningitis patients were administered anti-tubercular treatment. As per GOS, 38(85.4%) patients recovered with no neurological deficit. Six patients recovered with mild to moderate deficit. Conclusion: Low positive culture rates and inability to identify pathogens have been a diagnostic challenge faced by our team. Strong clinical suspicion, early empiric antibiotic and dexamethasone therapy perhaps contributed to very good recovery in our study.&#x0D; Keywords: Meningitis, Kuwait, Clinical Suspicion, Antibiotic, Dexamethasone

Epidemiological Characteristics of Hand, Foot, and Mouth Disease Outbreaks in Qingdao, 2009-2018.

Background:In China, hand, foot, and mouth disease (HFMD) outbreaks have become an important issue recent years. We analyzed the epidemiological characteristics of HFMD outbreaks in Qingdao during 2009–2018, and provided evidences for prevention and control of the disease.Methods:Data were analyzed by descriptive analysis and correlation analysis, and throat swabs were detected for enterovirus RNA using RT-PCR.Results:Overall, 116 HFMD outbreaks were reported in Qingdao during 2009–2018, with the epidemic of the outbreaks exhibiting a decreasing tendency. The characteristics of outbreaks presented two patterns, including two-peak pattern and rural area to urban-rural fringe area to urban areas pattern. Male patients were predominant in these outbreaks. The location of the outbreaks changed from nursery to community. Non-EV71/CA16 enteroviruses were gradually becoming predominant enteroviruses serotypes. The durations of outbreaks were positively correlated with response times and the number of cases.Conclusion:The epidemiological characteristics analysis of HFMD outbreaks could provide a scientific basis for the prevention and control the disease. Reporting and handling promptly are the keys to control epidemic outbreaks of HFMD.

The prevalence of enteroviral RNA and protein in mitral valves of chronic rheumatic heart disease.

Acute Rheumatic Fever/ Rheumatic Heart Disease (ARF/RHD), a sequel of group A streptococcal (GAS) infection, even today constitutes a public health issue in developing countries including India. Differences in the prevalence of ARF/RHD in countries with a similar prevalence of GAS infections indicate the role of other cofactors in pathogenesis of RHD. We investigated the prevalence of enterovirus (EV) in RHD by probing for both EV RNA and VP1 protein using Nonisotopic In Situ Hybridization (NISH) and Immunohistochemistry (IHC) respectively in 75 valvectomy specimens obtained from RHD cases. Twenty-eight (37%) of the valves showed tissue inflammation with lymphocytic infiltration in a majority of the cases. Twenty-six and 27 (38% and 40%) of the 68 valves showed the presence of EV by IHC and NISH respectively, indicating a very good association between the two tests; however, only about 46 to 48% of them exhibited tissue inflammation. In eight cases (12%) the EV genome was detectable in absence of VP1 protein perhaps indicating a latent viral infection. Due to a high degree of endemicity of EV in India, we are tempted to speculate that EV may be responsible for the severity and rapid progression of RHD. The virus could either be working synergistically with GAS or could be an opportunist infecting damaged valves.

Hand, foot and mouth disease associated with streptococcus infection in a child (clinical case)

In the presented clinical case, the combination of two pathogens (enterovirus and streptococcus) caused a severe and atypical course of the hand-foot-mouth syndrome in a child of primary school age. EVI from the first days of the disease represented by the typical localization of the primary elements of the rash and the characteristic intoxication syndrome was confirmed by the detection of enterovirus RNA in the feces. The course of the disease was undulating. In the first wave, skin rashes accompanied by soreness and itching corresponded to the classical EVI in terms of the nature of the elements, their localization and were combined with lesions of the epithelium of the oral mucosa. Starting from the 5th day of the disease, against the background of an increase in the severity of intoxication syndrome, bullous elements appeared on the skin of the extremities, an excess of the permissible values of antistreptolysin-O was found 12 times, and when sowing material from cracks in the corner of the mouth, Streptococcus pyogenes 106 KUO was isolated, which indicated a combination of the syndrome Hand-foot-mouth with streptococcal infection. Onychomadesis in the period of convalescence retrospectively testified to the dominant role of enterovirus in the presented clinical case.

Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback.

Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.

IRES-targeting small molecule inhibits enterovirus 71 replication via allosteric stabilization of a ternary complex

Enterovirus 71 (EV71) poses serious threats to human health, particularly in Southeast Asia, and no drugs or vaccines are available. Previous work identified the stem loop II structure of the EV71 internal ribosomal entry site as vital to viral translation and a potential target. After screening an RNA-biased library using a peptide-displacement assay, we identify DMA-135 as a dose-dependent inhibitor of viral translation and replication with no significant toxicity in cell-based studies. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein, thus repressing translation. This mechanism is supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.

Detection of Enteric Viruses and Bacterial Indicators in a Sewage Treatment Center and Shallow Water Bay.

The incidence of enteric viruses in treated wastewater and their potential release into the environment or use for agriculture are very critical matters in public health. In our study, PCR (polymerase chain reaction) analysis of enteric viruses was performed on 59 samples of influents and effluents collected from Tubli wastewater treatment plant (Water Pollution Control Center (WPCC)) and Tubli Bay, where the effluents were discharged, in Kingdom of Bahrain during two sampling periods. Four clinically essential waterborne enteric viruses were examined: enterovirus (EV), hepatitis A virus (HAV), astroviruses (AV), and rotaviruses (RV) and compared to standard bacterial and bacteriophages indicators of fecal pollution. Detection rates of EV, AV, HAV, and RV in the influent samples were 100%, 75%, 12.5%, and 12.5%, respectively, while 50% of the effluent samples from Tubli WPCC contained only EV RNA. None of the tested enteric viruses could be detected in any of the samples collected directly from Tubli Bay. Effluent samples from Tubli plant did not show significant seasonal differences. Since detection of enteric viruses genome does not necessarily indicate infectivity, the infectivity of these viruses was evaluated through isolation and growth of indictor bacteria and bacteriophages. High concentration of fecal bacteriological indicators was detected in all effluents samples (100%): 3.20 × 103 cfu/mL for E. coli, 1.32 × 103 cfu/mL for Salmonella spp., and 1.92 × 103 cfu/mL for Shigella spp. E. coli and Salmonella specific bacteriophages were also detected in the effluent samples in high titers. The combined results of PCR and bacterial enumeration point to a probable public health risk via the use of these wastewaters in agriculture or their discharge into the sea. Continuous surveillance of viral and bacterial prevalence and their resistance to sewage disinfection procedures could contribute to a better control of risks associated with the recycling of effluent wastewater and its release into the environment.

The RNA-dependent RNA polymerase of enterovirus A71 associates with ribosomal proteins and positively regulates protein translation

ABSTRACTEnteroviruses, which may cause neurological complications, have become a public health threat worldwide in recent years. Interactions between cellular proteins and enteroviral proteins could interfere with cellular biological processes to facilitate viral replication in infected cells. Enteroviral RNA-dependent RNA polymerase (RdRP), known as 3D protein, mainly functions as a replicase for viral RNA synthesis in infected cells. However, the 3D protein encoded by enterovirus A71 (EV-A71) could also interact with several cellular proteins to regulate cellular events and responses during infection. To globally investigate the functions of the EV-A71 3D protein in regulating biological processes in host cells, we performed immunoprecipitation coupled with liquid chromatography−tandem mass spectrometry (LC-MS/MS) to identify host proteins that may associate with the 3D protein. We found that the 3D protein interacts with factors involved in translation-related biological processes, including ribosomal proteins. In addition, polysome profiling analysis showed that the 3D protein cosediments with small and large subunits of ribosomes. We further discovered that the EV-A71 3D protein could enhance EV-A71 internal ribosome entry site (IRES)-dependent translation as well as cap-dependent translation. Collectively, this research demonstrated that the RNA polymerase encoded by EV-A71 could join a functional ribosomal complex and positively regulate viral and host translation.

Characteristics of Patients with Acute Flaccid Myelitis, United States, 2015-2018.

Observed peaks of acute flaccid myelitis (AFM) cases have occurred biennially since 2014 in the United States. We aimed to determine if AFM etiology differed between peak and nonpeak years, considering that clinical features of AFM differ by virus etiology. We compared clinical and laboratory characteristics of AFM cases that occurred during peak (2016 and 2018, n = 366) and nonpeak (2015 and 2017, n = 50) years. AFM patients in peak years were younger (5.2 years) than those in nonpeak years (8.3 years). A higher percentage of patients in peak years than nonpeak years had pleocytosis (86% vs. 60%), upper extremity involvement (33% vs. 16%), and an illness preceding limb weakness (90% vs. 62%) and were positive for enterovirus or rhinovirus RNA (38% vs. 16%). Enterovirus D68 infection was associated with AFM only in peak years. Our findings suggest AFM etiology differs between peak and nonpeak years.

Features of HIV infection in adults

Human immunodeficiency virus (HIV) infection remains a major medical problem due to its high prevalence and mortality. In order to study and identify clinical manifestations, frequency of their occurrence, as well as epidemiological features of this disease in the Penza region, we have analyzed 150 case histories. According to the results of the research, HIV infection is more common in men whose average age is 35.7 ± 9.1 years; in 69 % of cases-these are unemployed people; the average duration of the disease (by the date of registration at the dispensary) – 4.2 ± 4.1 years; unprotected sex is the leading factor of infection (35 %); there is a decrease in the share of infection by parenteral route with the use of drugs (32 %); in the structure of clinical manifestations, bronchopulmonary pathology (58 %) and chronic viral hepatitis C (60 %) prevails; diagnosed opportunistic infections were more often candidiasis of the oral cavity and esophagus (23 %), pneumocystic pneumonia (8 %) and Kaposi’s sarcoma (2 %). The etiological structure of purulent meningitis and meningoencephalitis is dominated by Staphylococcus aureus (25 %). The main etiological factor in the development of serous meningitis and meningoencephalitis are more often HSV DNA type 1, type 2 (12 %) and Enterovirus RNA (11 %).

Structure of the 5' Untranslated Region of Enteroviral Genomic RNA.

Enteroviral RNA genomes share a long, highly structured 5' untranslated region (5' UTR) containing a type I internal ribosome entry site (IRES). The 5' UTR is composed of stably folded RNA domains connected by unstructured RNA regions. Proper folding and functioning of the 5' UTR underlies the efficiency of viral replication and also determines viral virulence. We have characterized the structure of 5' UTR genomic RNA from coxsackievirus B3 using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and base-specific chemical probes in solution. Our results revealed novel structural features, including realignment of major domains, newly identified long-range interactions, and an intrinsically disordered connecting region. Together, these newly identified features contribute to a model for enteroviral 5' UTRs with type I IRES elements that links structure to function during the hierarchical processes directed by genomic RNA during viral infection.IMPORTANCE Enterovirus infections are responsible for human diseases, including myocarditis, pancreatitis, acute flaccid paralysis, and poliomyelitis. The virulence of these viruses depends on efficient recognition of the RNA genome by a large family of host proteins and protein synthesis factors, which in turn relies on the three-dimensional folding of the first 750 nucleotides of the molecule. Structural information about this region of the genome, called the 5' untranslated region (5' UTR), is needed to assist in the process of vaccine and antiviral development. This work presents a model for the structure of the enteroviral 5' UTR. The model includes an RNA element called an intrinsically disordered RNA region (IDRR). Intrinsically disordered proteins (IDPs) are well known, but correlates in RNA have not been proposed. The proposed IDRR is a 20-nucleotide region, long known for its functional importance, where structural flexibility helps explain recognition by factors controlling multiple functional states.

Detection and Characterization of Human Enteroviruses, Human Cosaviruses, and a New Human Parechovirus Type in Healthy Individuals in Osun State, Nigeria, 2016/2017.

Human enteroviruses and human parechoviruses are associated with a broad range of diseases and even severe and fatal conditions. For human cosaviruses, the etiological role is yet unknown. Little is known about the circulation of non-polio enteroviruses, human parechoviruses, and human cosaviruses in Nigeria. A total of 113 stool samples were collected from healthy individuals in Osun State between February 2016 and May 2017. RT-PCR assays targeting the 5′ non-coding region (5′ -NCR) were used to screen for human enteroviruses, human parechoviruses, and human cosaviruses. For human enteroviruses, species-specific RT-PCR assays targeting the VP1 regions were used for molecular typing. Inoculation was carried out on RD-A, CaCo-2, HEp-2C, and L20B cell lines to compare molecular and virological assays. Ten samples tested positive for enterovirus RNA with 11 strains detected, including CV-A13 (n = 3), E-18 (n = 2), CV-A20 (n = 1), CV-A24 (n = 1), EV-C99 (n = 1), and EV-C116 (n = 2). Three samples tested positive for human parechovirus RNA, and full genome sequencing on two samples allowed assignment to a new Parechovirus A type (HPeV-19). Thirty-three samples tested positive for cosavirus with assignment to species Cosavirus D and Cosavirus A based on the 5′-NCR region. Screening of stool samples collected from healthy individuals in Nigeria in 2016 and 2017 revealed a high diversity of circulating human enteroviruses, human parechoviruses, and human cosaviruses. Molecular assays for genotyping showed substantial benefits compared with those of cell-culture assays.

Pan-viral serology implicates enteroviruses in acute flaccid myelitis.

Since 2012, the United States has experienced a biennial spike in pediatric acute flaccid myelitis (AFM).1–6 Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF).2 We interrogated CSF from children with AFM (n=42) and pediatric other neurologic disease controls (n=58) for intrathecal anti-viral antibodies using a phage display library expressing 481,966 overlapping peptides derived from all known vertebrate and arboviruses (VirScan). We also performed metagenomic next-generation sequencing (mNGS) of AFM CSF RNA (n=20 cases), both unbiased and with targeted enrichment for EVs. Using VirScan, the only viral family significantly enriched by the CSF of AFM cases relative to controls was Picornaviridae, with the most enriched Picornaviridae peptides belonging to the genus Enterovirus (n=29/42 cases versus 4/58 controls). EV VP1 ELISA confirmed this finding (n=22/26 cases versus 7/50 controls). mNGS did not detect additional EV RNA. Despite rare detection of EV RNA, pan-viral serology identified frequently high levels of CSF EV-specific antibodies in AFM compared to controls, providing further evidence for a causal role of non-polio EVs in AFM.