Update on nonpolio enterovirus and parechovirus infections in neonates and young infants.

PURPOSE OF REVIEW: To review the epidemiology, clinical manifestations, and treatment strategies of nonpolio enterovirus and parechovirus (PeV) infections, and identify research gaps. RECENT FINDINGS: There is currently no approved antiviral agent for enterovirus or PeV infections, although pocapavir may be provided on a compassionate basis. Elucidation of the structure and functional features of enterovirus and PeV may lead to novel therapeutic strategies, including vaccine development. SUMMARY: Nonpolio human enterovirus and PeV are common childhood infections that are most severe among neonates and young infants. Although most infections are asymptomatic, severe disease resulting in substantial morbidity and mortality occurs worldwide and has been associated with local outbreaks. Long-term sequelae are not well understood but have been reported following neonatal infection of the central nervous system. The lack of antiviral treatment and effective vaccines highlight important knowledge gaps. Active surveillance ultimately may inform preventive strategies.

Surveillance To Track Progress Toward Poliomyelitis Eradication - Worldwide, 2021-2022.

Since the Global Polio Eradication Initiative (GPEI) was established in 1988, the number of wild poliovirus (WPV) cases has declined by >99.9%, and WPV serotypes 2 and 3 have been declared eradicated (1). By the end of 2022, WPV type 1 (WPV1) transmission remained endemic only in Afghanistan and Pakistan (2,3). However, during 2021-2022, Malawi and Mozambique reported nine WPV1 cases that were genetically linked to Pakistan (4,5), and circulating vaccine-derived poliovirus (cVDPV) outbreaks were detected in 42 countries (6). cVDPVs are oral poliovirus vaccine-derived viruses that can emerge after prolonged circulation in populations with low immunity allowing reversion to neurovirulence and can cause paralysis. Polioviruses are detected primarily through surveillance for acute flaccid paralysis (AFP), and poliovirus is confirmed through stool specimen testing. Environmental surveillance, the systematic sampling of sewage and testing for the presence of poliovirus, supplements AFP surveillance. Both surveillance systems were affected by the COVID-19 pandemic's effects on public health activities during 2020 (7,8) but improved in 2021 (9). This report updates previous reports (7,9) to describe surveillance performance during 2021-2022 in 34 priority countries.* In 2022, a total of 26 (76.5%) priority countries met the two key AFP surveillance performance indicator targets nationally compared with 24 (70.6%) countries in 2021; however, substantial gaps remain in subnational areas. Environmental surveillance expanded to 725 sites in priority countries, a 31.1% increase from the 553 sites reported in 2021. High-quality surveillance is critical to rapidly detect poliovirus transmission and enable prompt poliovirus outbreak response to stop circulation. Frequent monitoring of surveillance guides improvements to achieve progress toward polio eradication.

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OBJECTIVES: To investigate the positive rate of enterovirus (EV) nucleic acid in throat swabs of term late neonates hospitalized during the coronavirus disease 2019 (COVID-19) epidemic and the clinical characteristics of the neonates. METHODS: A single-center cross-sectional study was performed on 611 term late infants who were hospitalized in the neonatal center from October 2020 to September 2021. Throat swabs were collected on admission for coxsackie A16 virus/EV71/EV universal nucleic acid testing. According to the results of EV nucleic acid test, the infants were divided into a positive EV nucleic acid group (8 infants) and a negative EV nucleic acid group (603 infants). Clinical features were compared between the two groups. RESULTS: Among the 611 neonates, 8 tested positive for EV nucleic acid, with a positive rate of 13.1‰, among whom 7 were admitted from May to October. There was a significant difference in the proportion of infants contacting family members with respiratory infection symptoms before disease onset between the positive and negative EV nucleic acid groups (75.0% vs 10.9%, P<0.001). There were no significant differences between the two groups in demographic data, clinical symptoms, and laboratory test results (P>0.05). CONCLUSIONS: There is a certain proportion of term late infants testing positive for EV nucleic acid in throat swabs during the COVID-19 epidemic, but the proportion is low. The clinical manifestations and laboratory test results of these infants are non-specific. Transmission among family members might be an important cause of neonatal EV infection.

Evaluation of 3D Human Intestinal Organoids as a Platform for EV-A71 Antiviral Drug Discovery.

Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2'-C-methylcytidine (2'CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies.

Optimization and performance evaluation of an automated filtration method for the recovery of SARS-CoV-2 and other viruses in wastewater.

A rapid virus concentration method is needed to get high throughput. Reliable results of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection in wastewater are necessary for applications in wastewater-based epidemiology. In this study, an automated filtration method using a concentrating pipette (CP Select; Innovaprep) was applied to detect SARS-CoV-2 in wastewater samples with several modifications to increase its sensitivity and throughput. The performance of the CP Select method was compared to other concentration methods (polyethylene glycol precipitation and direct capture using silica column) to evaluate its applicability to SARS-CoV-2 detection in wastewater. SARS-CoV-2 RNA was successfully detected in six of eight wastewater samples using the CP Select method, whereas other methods could detect SARS-CoV-2 RNA in all wastewater samples. Enteric viruses, such as noroviruses of genogroups I (NoVs-GI) and II (NoVs-GII) and enteroviruses, were tested, resulting in 100 % NoVs-GII detection using all concentration methods. As for NoVs-GI and enteroviruses, all methods gave comparable number of detected samples in wastewater samples. This study showed that the optimized CP Select method was less sensitive in SARS-CoV-2 detection in wastewater than other methods, whereas all methods were applicable to detect or recover other viruses in wastewater.

The 2022 outbreak and the pathobiology of the coxsackie virus [hand foot and mouth disease] in India.

Outbreaks of HFMD in children aged <5 years have been reported worldwide and the major causative agents are Coxsackievirus (CV) A16, enterovirus (EV)-A71 and recently CVA6. In India, HFMD is a disease that is not commonly reported. The purpose of the study was to identify the enterovirus type(s) associated with large outbreak of Hand, foot, and mouth disease during COVID-19 pandemic in 2022. Four hundred and twenty five clinical samples from 196-suspected cases were collected from different parts of the country. This finding indicated the emergence of CVA6 in HFMD along with CVA16, soon after the gradual easing of non-pharmaceutical interventions during-pandemic COVID-19 and the relevance of continued surveillance of circulating enterovirus types in the post-COVID pandemic era.

Genotype Diversity of Enteric Viruses in Wastewater Amid the COVID-19 Pandemic.

Viruses remain the leading cause of acute gastroenteritis (AGE) worldwide. Recently, we reported the abundance of AGE viruses in raw sewage water (SW) during the COVID-19 pandemic, when viral AGE patients decreased dramatically in clinics. Since clinical samples were not reflecting the actual state, it remained important to determine the circulating strains in the SW for preparedness against impending outbreaks. Raw SW was collected from a sewage treatment plant in Japan from August 2018 to March 2022, concentrated by polyethylene-glycol-precipitation method, and investigated for major gastroenteritis viruses by RT-PCR. Genotypes and evolutionary relationships were evaluated through sequence-based analyses. Major AGE viruses like rotavirus A (RVA), norovirus (NoV) GI and GII, and astrovirus (AstV) increased sharply (10-20%) in SW during the COVID-19 pandemic, though some AGE viruses like sapovirus (SV), adenovirus (AdV), and enterovirus (EV) decreased slightly (3-10%). The prevalence remained top in the winter. Importantly, several strains, including G1 and G3 of RVA, GI.1 and GII.2 of NoV, GI.1 of SV, MLB1 of AstV, and F41 of AdV, either emerged or increased amid the pandemic, suggesting that the normal phenomenon of genotype changing remained active over this time. This study crucially presents the molecular characteristics of circulating AGE viruses, explaining the importance of SW investigation during the pandemic when a clinical investigation may not produce the complete scenario.

Current status of hand-foot-and-mouth disease.

Hand-foot-and-mouth disease (HFMD) is a viral illness commonly seen in young children under 5 years of age, characterized by typical manifestations such as oral herpes and rashes on the hands and feet. These symptoms typically resolve spontaneously within a few days without complications. Over the past two decades, our understanding of HFMD has greatly improved and it has received significant attention. A variety of research studies, including epidemiological, animal, and in vitro studies, suggest that the disease may be associated with potentially fatal neurological complications. These findings reveal clinical, epidemiological, pathological, and etiological characteristics that are quite different from initial understandings of the illness. It is important to note that HFMD has been linked to severe cardiopulmonary complications, as well as severe neurological sequelae that can be observed during follow-up. At present, there is no specific pharmaceutical intervention for HFMD. An inactivated Enterovirus A71 (EV-A71) vaccine that has been approved by the China Food and Drug Administration (CFDA) has been shown to provide a high level of protection against EV-A71-related HFMD. However, the simultaneous circulation of multiple pathogens and the evolution of the molecular epidemiology of infectious agents make interventions based solely on a single agent comparatively inadequate. Enteroviruses are highly contagious and have a predilection for the nervous system, particularly in child populations, which contributes to the ongoing outbreak. Given the substantial impact of HFMD around the world, this Review synthesizes the current knowledge of the virology, epidemiology, pathogenesis, therapy, sequelae, and vaccine development of HFMD to improve clinical practices and public health efforts.

Prevalence of Non-Polio Enteroviruses in the Sewage of Guangzhou City, China, from 2013 to 2021.

Continuous surveillance of enteroviruses (EVs) in urban domestic sewage can timely reflect the circulation of EVs in the environment and crowds, and play a predictive and early warning role in EV-related diseases. To better understand the long-term epidemiological trends of circulating EVs and EV-related diseases, we conducted a 9-year (2013 to 2021) surveillance study of non-polio EVs (NPEVs) in urban sewage in Guangzhou city, China. After concentrating and isolating the viruses from the sewage samples, NPEVs were detected and molecular typing was performed. Twenty-one different NPEV serotypes were identified. The most isolated EVs were echovirus 11 (E11), followed by coxsackievirus (CV) B5, E6, and CVB3. EV species B prevailed in sewage samples, but variations in the annual frequency of different serotypes were also observed in different seasons, due to spatial and temporal factors. E11 and E6 were detected continuously before 2017, and the number of isolates was relatively stable during the surveillance period. However, after their explosive growth in 2018 and 2019, their numbers suddenly decreased significantly. CVB3 and CVB5 had alternating trends; CVB5 was most frequently detected in 2013 to 2014 and 2017 to 2018, while CVB3 was most frequently detected in 2015 to 2016 and 2020 to 2021. Phylogenetic analysis showed that at least two different transmission chains of CVB3 and CVB5 were prevalent in Guangzhou City. Our results show that in the absence of a comprehensive and systematic EV-related disease surveillance system in China, environmental surveillance is a powerful and effective tool to strengthen and further investigate the invisible transmission of EVs in the population. IMPORTANCE This study surveilled urban sewage samples from north China for 9 years to monitor enteroviruses. Samples were collected, processed, and viral identification and molecular typing were performed. We detected 21 different non-polio enteroviruses (NPEVs) with yearly variations in prevalence and peak seasons. In addition, this study is very important for understanding the epidemiology of EVs during the COVID-19 pandemic, as the detection frequency and serotypes of EVs in sewage changed considerably around 2020. We believe that our study makes a significant contribution to the literature because our results strongly suggest that environmental surveillance is an exceptionally important tool, which can be employed to detect and monitor organisms of public health concern, which would otherwise be missed and under-reported by case-based surveillance systems alone.

Increased detection of Echovirus 6-associated meningitis in patients hospitalized during the COVID-19 pandemic, Israel 2021-2022.

BACKGROUND: Outbreaks of enteroviral meningitis occur periodically and may lead to hospitalization and severe disease. OBJECTIVE: To analyze and describe the meningitis outbreak in patients hospitalized in Israel in 2021-2022, during the COVID-19 pandemic. RESULTS: In December 2021, before the emergence of the SARS-CoV-2 omicron variant, an off-season increase in enterovirus (EV) infections was observed among patients hospitalized with meningitis. In January 2022, enterovirus cases decreased by 66% in parallel with the peak of the Omicron wave, and then increased rapidly by 78% in March (compared with February) after a decline in Omicron cases. Sequencing of the enterovirus-positive samples showed a dominance of echovirus 6 (E-6) (29%) before and after the Omicron wave. Phylogenetic analysis found that all 29 samples were very similar and all clustered in the E-6 C1 subtype. The main E-6 symptoms observed were fever and headache, along with vomiting and neck stiffness. The median patient age was 25 years, with a broad range (0-60 years). CONCLUSION: An upsurge in enterovirus cases was observed after the decline of the SARS-CoV-2 omicron wave. The dominant subtype was E-6, which was present prior to the emergence of the omicron variant, but increased rapidly only after the omicron wave decline. We hypothesize that the omicron wave delayed the rise in E-6-associated meningitis.

Monitoring Enteroviruses and SARS-CoV-2 in Wastewater Using the Polio Environmental Surveillance System in Japan.

In the global strategy for polio eradication, environmental surveillance (ES) has been established worldwide to monitor polioviruses. In addition, nonpolio enteroviruses are simultaneously isolated from wastewater under this ES program. Hence, ES can be used to monitor enteroviruses in sewage to supplement clinical surveillance. In response to the coronavirus disease 2019 (COVID-19) pandemic, we also monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage using the polio ES system in Japan. Enterovirus and SARS-CoV-2 were detected in sewage from January 2019 to December 2021 and from August 2020 to November 2021, respectively. Enterovirus species such as echoviruses and coxsackieviruses were frequently detected by ES in 2019, indicating the circulation of these viruses. After the onset of the COVID-19 pandemic, sewage enterovirus detection and related patient reports were notably reduced in 2020 to 2021, suggesting changes in the hygiene behaviors of the human population in response to the pandemic. Our comparative experiment with a total of 520 reverse transcription-quantitative PCR (RT-qPCR) assays for SARS-CoV-2 detection demonstrated that the solid-based method had a significantly higher detection rate than that of the liquid-based method (24.6% and 15.9%, respectively). Moreover, the resulting RNA concentrations were correlated with the number of new COVID-19 cases (Spearman's r = 0.61). These findings indicate that the existing polio ES system can be effectively used for enterovirus and SARS-CoV-2 sewage monitoring using different procedures such as virus isolation and molecular-based detection. IMPORTANCE Long-term efforts are required to implement surveillance programs for the ongoing COVID-19 pandemic, and they will be required even in the postpandemic era. We adopted the existing polio environmental surveillance (ES) system for SARS-CoV-2 sewage monitoring in Japan as a practical and cost-effective approach. Moreover, the ES system routinely detects enteroviruses from wastewater and, therefore, can be used for enterovirus monitoring. The liquid fraction of the sewage sample is used for poliovirus and enterovirus detection, and the solid fraction can be used for SARS-CoV-2 RNA detection. The present study demonstrates how the existing ES system can be used for monitoring enteroviruses and SARS-CoV-2 in sewage.

The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D.

RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses.

Employing functionalized graphene quantum dots to combat coronavirus and enterovirus.

The ongoing COVID-19 (i.e., coronavirus) pandemic continues to adversely affect the human life, economy, and the world's ecosystem. Although significant progress has been made in developing antiviral materials for the coronavirus, much more work is still needed. In this work, N-functionalized graphene quantum dots (GQDs) were designed and synthesized as the antiviral nanomaterial for Feline Coronavirus NTU156 (FCoV NTU156) and Enterovirus 71 (EV71)) with ultra-high inhibition (>99.9%). To prepare the GQD samples, a unique solid-phase microwave-assisted technique was developed and the cell toxicity was established on the H171 and H184 cell lines after 72 h incubation, indicating superior biocompatibility. The surface functionality of GQDs (i.e., the phenolic and amino groups) plays a vital role in interacting with the receptor-binding-domain of the spike protein. It was also found that the addition of polyethylene glycol is advantageous for the dispersion and the adsorption of functionalized GQDs onto the virus surface, leading to an enhanced virus inhibition. The functionality of as-prepared GQD nanomaterials was further confirmed where a functionalized GQD-coated glass was shown to be extremely effective in hindering the virus spread for a relatively long period (>20 h).

Circulation of Rhinoviruses and/or Enteroviruses in Pediatric Patients With Acute Respiratory Illness Before and During the COVID-19 Pandemic in the US.

Importance: Rhinoviruses and/or enteroviruses, which continued to circulate during the COVID-19 pandemic, are commonly detected in pediatric patients with acute respiratory illness (ARI). Yet detailed characterization of rhinovirus and/or enterovirus detection over time is limited, especially by age group and health care setting. Objective: To quantify and characterize rhinovirus and/or enterovirus detection before and during the COVID-19 pandemic among children and adolescents seeking medical care for ARI at emergency departments (EDs) or hospitals. Design, Setting, and Participants: This cross-sectional study used data from the New Vaccine Surveillance Network (NVSN), a multicenter, active, prospective surveillance platform, for pediatric patients who sought medical care for fever and/or respiratory symptoms at 7 EDs or hospitals within NVSN across the US between December 2016 and February 2021. Persons younger than 18 years were enrolled in NVSN, and respiratory specimens were collected and tested for multiple viruses. Main Outcomes and Measures: Proportion of patients in whom rhinovirus and/or enterovirus, or another virus, was detected by calendar month and by prepandemic (December 1, 2016, to March 11, 2020) or pandemic (March 12, 2020, to February 28, 2021) periods. Month-specific adjusted odds ratios (aORs) for rhinovirus and/or enterovirus-positive test results (among all tested) by setting (ED or inpatient) and age group (<2, 2-4, or 5-17 years) were calculated, comparing each month during the pandemic to equivalent months of previous years. Results: Of the 38 198 children and adolescents who were enrolled and tested, 11 303 (29.6%; mean [SD] age, 2.8 [3.7] years; 6733 boys [59.6%]) had rhinovirus and/or enterovirus-positive test results. In prepandemic and pandemic periods, rhinoviruses and/or enteroviruses were detected in 29.4% (9795 of 33 317) and 30.9% (1508 of 4881) of all patients who were enrolled and tested and in 42.2% (9795 of 23 236) and 73.0% (1508 of 2066) of those with test positivity for any virus, respectively. Rhinoviruses and/or enteroviruses were the most frequently detected viruses in both periods and all age groups in the ED and inpatient setting. From April to September 2020 (pandemic period), rhinoviruses and/or enteroviruses were detectable at similar or lower odds than in prepandemic years, with aORs ranging from 0.08 (95% CI, 0.04-0.19) to 0.76 (95% CI, 0.55-1.05) in the ED and 0.04 (95% CI, 0.01-0.11) to 0.71 (95% CI, 0.47-1.07) in the inpatient setting. However, unlike some other viruses, rhinoviruses and/or enteroviruses soon returned to prepandemic levels and from October 2020 to February 2021 were detected at similar or higher odds than in prepandemic months in both settings, with aORs ranging from 1.47 (95% CI, 1.12-1.93) to 3.01 (95% CI, 2.30-3.94) in the ED and 1.36 (95% CI, 1.03-1.79) to 2.44 (95% CI, 1.78-3.34) in the inpatient setting, and in all age groups. Compared with prepandemic years, during the pandemic, rhinoviruses and/or enteroviruses were detected in patients who were slightly older, although most (74.5% [1124 of 1508]) were younger than 5 years. Conclusions and Relevance: Results of this study show that rhinoviruses and/or enteroviruses persisted and were the most common respiratory virus group detected across all pediatric age groups and in both ED and inpatient settings. Rhinoviruses and/or enteroviruses remain a leading factor in ARI health care burden, and active ARI surveillance in children and adolescents remains critical for defining the health care burden of respiratory viruses.

Drug Repositioning for Hand, Foot, and Mouth Disease.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease in children caused by a group of enteroviruses. HFMD currently presents a major threat to infants and young children because of a lack of antiviral drugs in clinical practice. Drug repositioning is an attractive drug discovery strategy aimed at identifying and developing new drugs for diseases. Notably, repositioning of well-characterized therapeutics, including either approved or investigational drugs, is becoming a potential strategy to identify new treatments for virus infections. Various types of drugs, including antibacterial, cardiovascular, and anticancer agents, have been studied in relation to their therapeutic potential to treat HFMD. In this review, we summarize the major outbreaks of HFMD and the progress in drug repositioning to treat this disease. We also discuss the structural features and mode of action of these repositioned drugs and highlight the opportunities and challenges of drug repositioning for HFMD.

Disruption of seasonal enterovirus and parechovirus detections in the CSF and plasma of children during the COVID-19 pandemic.

BACKGROUND: Enteroviruses (EV) and parechovirus (PeV) are a common cause of CNS infection in children. Both viruses demonstrate consistent seasonal patterns, with detections mainly in the summer-fall months. While research has shown COVID-19 pandemic-related disruption of traditional seasonality of respiratory pathogens, the pandemic's impact on non-respiratory pathogens is less understood. The aim of this study was to quantify the EV/PeV seasonal variations during pre-COVID years compared to variations observed during the COVID pandemic. METHODS: Patients with EV/PeV testing of CSF/plasma between January 2012 through September 2022 were identified. Restricted cubic spline methods were used to model the detections. Poisson models were utilized to model pre-COVID (2012-2019) EV/PeV detections. The expected seasonal trends from these models were then compared to the observed EV/PeV detections during the COVID pandemic (2020-2022). RESULTS: A total of 5199 patients were included. The annual pre-pandemic proportion of EV detections ranged between 7.5%-20.3%. PeV exhibited a biennial pattern with peak proportions between 8.0%-16.3%. EV/PeV detections during the COVID pandemic period, especially during 2020 and 2021, were considerably lower than would have been expected based on pre-pandemic modeling. However, PeV detections from January through September 2022 nearly reached the pre-pandemic modeled expectation, including instances of exceeded expectations. CONCLUSIONS: A significant disruption in expected seasonal EV/PeV detections was observed during the early phases of the COVID-19 pandemic. However, testing that occurred during summer-fall of 2022, when social mitigation initiatives were relaxed, showed a rapid increase in detections. Additional data are needed to further understand which public health initiatives are effective at decreasing EV/PeV transmission.

EV-A71 induced IL-1ß production in THP-1 macrophages is dependent on NLRP3, RIG-I, and TLR3.

Enterovirus A71 (EV-A71) is an emerging enterovirus that can cause neurological complications. Enhanced serum IL-1ß levels were observed in EV-A71 patients with severe neurological symptoms. However, the roles of sensors in enterovirus-induced IL-1ß production are unclear. In this study, we identified that pattern recognition receptors, including RIG-I, TLR3, and TLR8, are implicated in EV-A71-triggered IL-1ß release in human macrophages. EV-A71 infection results in caspase-1 and caspase-8, which act as regulators of EV-A71-induced NLRP3 and RIG-I inflammasome activation. Moreover, knockdown of the expression of TLR3 and TLR8 decreased the released IL-1ß in an NLRP3-dependent manner. Since TLR3 and TLR8 ligands promote NLRP3 inflammasome activation via caspase-8, the alternative pathway may be involved. In summary, these results indicate that activation of the NLRP3 and RIG-I inflammasomes in EV-A71-infected macrophages is mediated by caspase-1 and caspase-8 and affected by TLRs, including TLR3 and TLR8.

Identification of a neutralizing linear epitope within the VP1 protein of coxsackievirus A10.

BACKGROUND: Coxsackievirus A10 (CV-A10) is a leading cause of hand, foot, and mouth disease (HFMD). It is necessary to identify neutralizing epitopes to investigate and develop an epitope-based vaccine against CV-A10. The viral protein VP1 is the immunodominant capsid protein and contains the critical neutralizing epitope. However, neutralizing epitopes within VP1 protein of CV-A10 have not been well characterized. METHODS: Bioinformatics techniques were applied to predict linear epitopes on the CV-A10 VP1 protein. The advanced structural features of epitopes were analyzed by three-dimensional (3D) modeling. The anticipated epitope peptides were synthesized and used to immunize mice as antigens. ELISA and micro-neutralization assay were used to determine the specific IgG antibody and neutralizing antibody titers. The protective efficacy of the epitope peptides in vivo was evaluated using a passive immunization/challenge assay. RESULTS: Three linear epitopes (EP3, EP4, and EP5) were predicted on CV-A10 VP1, all spatially exposed on the capsid surface, and exhibited adequate immunogenicity. However, only EP4, corresponding to residues 162-176 of VP1, demonstrated potent neutralization against CV-A10. To determine the neutralizing capacity of EP4 further, EP4 double-peptide was synthesized and injected into mice. The mean neutralizing antibody titer of the anti-EP4 double-peptide sera was 1:50.79, which provided 40% protection against lethal infection with CV-A10 in neonatal mice. In addition, sequence and advanced structural analysis revealed that EP4 was highly conserved among representative strains of CV-A10 and localized in the EF loop region of VP1, like EV-A71 SP55 or CV-A16 PEP55. CONCLUSIONS: These data demonstrate that EP4 is a specific linear neutralizing epitope on CV-A10 VP1. Its protective efficacy can be enhanced by increasing its copy number, which will be the foundation for developing a CV-A10 epitope-based vaccine.