EV-A71 Infection Research Articles

Two cases of hand, foot and mouth disease caused by enterovirus A71 after vaccination

BackgroundEnterovirus A71 (EVA71) is one of the main pathogens causing hand, foot and mouth disease (HFMD). In China, the proportion of cases of HFMD caused by EVA71 is known to be significantly lower following EVA71 vaccination; however, infection with EVA71 can still occur after vaccination. MethodsThe complete genomic sequences of EVA71-KM18A and KM18B (from two rare cases of EVA71 infection following vaccination) were obtained. Phylogenetic analysis, nucleotide mutation analysis, recombinant analysis and comparative analysis of amino acid mutations were performed. ResultsPhylogenetic analysis determined that the EVA71 strains belonged to the C4a subgenotype. The KM18A and KM18B strains were highly similar to the vaccine strains. For the KM18B strain, there were some obvious homologous recombination signals in the 5’non-coding region, region 2A, region 2C and region 3D. Amino acid mutations were observed in the SP55 (position 729) and 71-6 (position 500) conformational neutralizing epitopes of the KM18A and KM18B strains. ConclusionsThese amino acid mutations may affect the SP55 and 71-6 conformational neutralizing epitopes and change their spatial conformation, thereby weakening vaccine effectiveness.

Circulation of enterovirus A71 during 2019–2020, Marseille, France

Enteroviruses A71 (EVs-A71) are known to cause serious neurological infections, especially in the pediatric population. We report here eight cases of EV-A71 infection diagnosed in Marseille over the past 2 years (seven cases in 2019 and one case in 2020). Only children under 5 years of age were affected, including one case of acute flaccid paralysis. Viral RNA was detected by RT-PCR in peripheral samples for all cases (feces and upper respiratory samples). Phylogenetic analyses based on VP1 and 2C3C coding regions revealed that all these cases of EV-A71 infection were caused by viruses belonging to the subgenogroup C1 that currently circulates in Europe and that these viruses are genetically closed to other EVs-A71 recently detected in European countries. These data therefore reinforce the usefulness of the enterovirus surveillance network and the need for systematic screening for EV-A71 in case of an enteroviral infection. This study therefore suggests that the systematic screening for EV-A71 in case of enteroviral infection could provide additional data for enterovirus surveillance networks.

Molecular determinants and heterogeneity underlying host response to EV-A71 infection at single-cell resolution

ABSTRACT The pathogenic human enterovirus EV-A71 has raised serious public health concerns. A hallmark of EV-A71 infection is the distortion of host transcriptomes in favour of viral replication. While high-throughput approaches have been exploited to dissect these gene dysregulations, they do not fully capture molecular perturbations at the single-cell level and in a physiologically relevant context. In this study, we applied a single-cell RNA sequencing approach on infected differentiated enterocyte cells (C2BBe1), which model the gastrointestinal epithelium targeted initially by EV-A71. Our single-cell analysis of EV-A71-infected culture provided several lines of illuminating observations: 1) This systems approach demonstrated extensive cell-to-cell variation in a single culture upon viral infection and delineated transcriptomic differences between the EV-A71-infected and bystander cells. 2) By analysing expression profiles of known EV-A71 receptors and entry facilitation factors, we found that ANXA2 was closely correlated in expression with the viral RNA in the infected population, supporting its role in EV-A71 entry in the enteric cells. 3) We further catalogued dysregulated lncRNAs elicited by EV-A71 infection and demonstrated the functional implication of lncRNA CYTOR in promoting EV-A71 replication. Viewed together, our single-cell transcriptomic analysis illustrated at the single-cell resolution the heterogeneity of host susceptibility to EV-A71 and revealed the involvement of lncRNAs in host antiviral response.

Early-life EV-A71 infection augments allergen-induced airway inflammation in asthma through trained macrophage immunity.

Virus-induced asthma is prevalent among children, but its underlying mechanisms are unclear. Accumulated evidence indicates that early-life respiratory virus infection increases susceptibility to allergic asthma. Nonetheless, the relationship between systemic virus infections, such as enterovirus infection, and the ensuing effects on allergic asthma development is unknown. Early-life enterovirus infection was correlated with higher risks of allergic diseases in children. Adult mice exhibited exacerbated mite allergen-induced airway inflammation following recovery from EV-A71 infection in the neonatal period. Bone marrow-derived macrophages (BMDMs) from recovered EV-A71-infected mice showed sustained innate immune memory (trained immunity) that could drive naïve T helper cells toward Th2 and Th17 cell differentiation when in contact with mites. Adoptive transfer of EV-A71-trained BMDMs induced augmented allergic inflammation in naïve recipient mice, which was inhibited by 2-deoxy-D-glucose (2-DG) pretreatment, suggesting that trained macrophages following enterovirus infection are crucial in the progression of allergic asthma later in life.

A practical method for evaluating the in vivo efficacy of EVA-71 vaccine using a hSCARB2 knock-in mouse model

Hand-foot-and-mouth disease is a contagious disease common among children under 5 years old worldwide. It is caused by strains of enterovirus, especially EV-A71, which can lead to severe disease. Vaccines are the only way to fight this disease. Accordingly, it is necessary to establish an efficient and accurate methodology to evaluate vaccine efficacy in vivo. Here, we established a practical method using a hSCARB2 knock-in mouse model, which was susceptible to EV-A71 infection at 5-6 weeks of age, to directly determine the efficacy of vaccines. Unlike traditional approaches, one-week old hSCARB2 mice were immunized twice with a licensed vaccine, with an interval of a week. The titer of antibodies was measured after one week. Mice at four weeks of age were challenged with EV-A71 intraperitoneally and intracranially, respectively. The unimmunized hSCARB2 mice displayed systemic clinical symptoms and succumbed to the disease at a rate of approximately 50%. High viral loads were detected in the lungs, brain and muscles, accompanied by clear pathological changes. The expression of IL-1β, IL-13, IL-17 and TNF-α was significantly upregulated. By contrast, the immunized group was practically normal and indistinguishable from the control mice. These results indicate that the hSCARB2 knock-in mouse is susceptible to infection in adulthood, and the in vivo efficacy of EV-A71 vaccine could be directly evaluated in this mouse model. The method developed here may be used in the development of new vaccines against HFMD or quality control of licensed vaccines.

Incidence of Infection of Enterovirus 71 and Coxsackieviruses A6 and A16 among Household Contacts of Index Cases in Dong Thap Province, Southern Vietnam.

Background Scarce information exists about immunity to hand, foot, and mouth disease (HFMD) among household contacts of index cases in Vietnam and what that means for reducing ongoing HFMD transmission in the community. Methods We analyzed neutralizing antibodies (NT) and the incidence of enterovirus (EVs) infection among household contacts of index cases in a province where HFMD remains endemic. Throat swab and 2 mL blood samples from household contacts were collected at enrollment, during and after 2 weeks follow-up. Results The incidence of EV-A71 infection among household contacts was 40/84 (47.6%, 95% Cl: 36.9-58.3%), compared with 106/336 (31.5%, 95% Cl: 26.6-36.5%) for CV-A6 and 36/107 (33.6%, 95% Cl: 24.7-42.6%) for CV-A16. The incidence of CV-A6 infection was fairly constant across ages; in contrast, CV-A71 and CV-A16 had some variation across ages. At baseline, higher geometric mean titer (GMT) of EV-A71, CV-A6, and CV-A16 antibody titers was found for 25-34-year groups (range 216.3 to 305.0) compared to the other age groups. There was a statistically significant difference in GMT values of CV-A6 and CV-A16 between those who had an infection or did not have infection among households with an index case of these serotypes. Conclusions Our results indicated that adults were becoming infected with HFMD and could be contributing to the transmission. There is, therefore, a need for considering the household setting as an additional target for intervention programs for HFMD.

Transcriptome analysis following enterovirus 71 and coxsackievirus A16 infection in respiratory epithelial cells.

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major pathogens responsible for hand, foot and mouth disease (HFMD), but the mechanism by which these viruses cause disease remains unclear. In this study, we used transcriptome sequencing technology to investigate changes in the transcriptome profiles after infection with EV-A71 and CV-A16 in human bronchial epithelial (16HBE) cells. Using systematic bioinformatics analysis, we then searched for useful clues regarding the pathogenesis of HFMD. As a result, a total of 111 common differentially expressed genes were present in both EV-A71- and CV-A16-infected cells. A trend analysis of these 111 genes showed that 91 of them displayed the same trend in EV-A71 and CV-A16 infection, including 49 upregulated genes and 42 downregulated genes. These 91 genes were further used to conduct GO, pathway, and coexpression network analysis. It was discovered that enriched GO terms (such as histone acetylation and positive regulation of phosphorylation) and pathways (such as glycosylphosphatidylinositol (GPI)-anchor biosynthesis and DNA replication) might be closely associated with the pathogenic mechanism of these two viruses, and key genes (such as TBCK and GPC) might be involved in the progression of HFMD. Finally, we randomly selected 10 differentially expressed genes for qRT-PCR to validate the transcriptome sequencing data. The experimental qRT-PCR results were roughly in agreement with the results of transcriptome sequencing. Collectively, our results provide clues to the mechanism of pathogenesis of HFMD induced by EV-A71 and CV-A16.Electronic supplementary materialThe online version of this article (10.1007/s00705-020-04821-1) contains supplementary material, which is available to authorized users.

Epidemiological characteristics of fatal cases of hand, foot, and mouth disease in children under 5 years old in China, 2008-2018

Objective: To understand the epidemiological characteristics and risk factors of fatal cases of hand, foot, and mouth disease (HFMD) in children under 5 years old in China from 2008 to 2018, and provide evidence for the development of targeted prevention and control measures and reduction of the incidence of fatal HFMD cases. Methods: The incidence data of reported HFMD cases in China during 2008-2018 were collected from the National Notifiable Disease Surveillance Reporting System of China for the analyses on the demographic characteristics, spatial distribution, diagnosis or reporting and pathogen spectrum of the HFMD cases. Then the risk factors causing deaths were analyzed by using logistic regression model. Results: From 2008 to 2018, a total of 3 646 fatal cases of HFMD in children under 5 years old were reported in China. There were more fatal HFMD cases in boys than in girls (1.82∶1), the death mainly occurred in age group 0 to 2 years (87.71%). Adjusted mortality rate of HFMD in children under 5 years old in China declined from 0.87 per 100 000 in 2010 to 0.11 per 100 000 in 2018 (APC=-23.20%). In the 2 523 laboratory-confirmed deaths, 2 323 (92.07%) were EV-A71 infections, but the constituents of CV-A16 and other enterovirus infections increased. The interval from onset to diagnosis M=2(P(25)-P(75):2-4)d. The interval from onset to death M=3(P(25)-P(75):2-4)d. Age between 0 and 1 years, EV-A71 infection, longer interval between onset and diagnosis, and living in rural area were the risk factors causing fatal HFMD cases. Conclusions: The number of the fatal cases, the rate of mortality and case fatality HFMD in China had shown downward trends since 2010. EV-A71 is still the main pathogen causing fatal cases, but we should pay more attention to gene pattern of the other enteroviruses except EV-A71 and CV-A16. To reduce the risk of the fatal cases we should strengthen the health education about the immunization of EV-A71 inactivated vaccines and reduce the interval from onset to diagnosis in young children in western provinces and rural areas.

Exosomes Facilitate Transmission of Enterovirus A71 From Human Intestinal Epithelial Cells.

BackgroundEnterovirus A71 (EV-A71) has been noted for its tendency to lead to neurological manifestations in young children and infants. Although the alimentary tract has been identified as the primary replication site of this virus, how EV-A71 replicates in the gut and is transmitted to other organs remains unclear.MethodsBy using differentiated C2BBe1 cells as a model, we observed that intestinal epithelial cells (IECs) were permissive to EV-A71 infection, and viral particles were released in a nonlytic manner.ResultsThe coexistence of active caspase 3 and EV-A71 protein was observed in the infected undifferentiated C2BBe1 and RD cells but not in the infected differentiated C2BBe1 cells. Furthermore, EV-A71 infection caused differentiated C2BBe1 and intestinal organoids to secrete exosomes containing viral components and have the ability to establish active infection. Inhibition of the exosome pathway decreased EV-A71 replication and release in IECs and increased the survival rates of infected animals.ConclusionsOur findings showed that EV-A71 is able to be actively replicated in enterocytes, and that the exosome pathway is involved in the nonlytic release of viral particles, which may be useful for developing antiviral strategies.

Antiviral activity of silymarin in comparison with baicalein against EV-A71

BackgroundThe hand, foot and mouth disease (HFMD) is a febrile and exanthematous childhood disease mainly caused by Enterovirus 71 (EV-A71). In severe HFMD, virulent EV-A71 strains can cause acute flaccid paralysis and cardiopulmonary edema leading to death. Currently, no FDA approved antiviral treatment or vaccine is available for EV-A71. Flavonoids such as silymarin and baicalein are known to possess in vitro antiviral properties against viruses. In this study, the cytotoxicity and antiviral activity of silymarin, baicalein and baicalin were investigated.MethodsThe cytotoxic effects of three flavonoids towards rhabdomyosarcoma (RD) cells were first examined using cell proliferation MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Compounds found to be non-cytotoxic in RD cells were evaluated for their in vitro antiviral properties against the EV-A71 subgenotype B4 strain 41 (5865/SIN/000009) using antiviral assays. Viral infectivity was determined by reduction of the formation of plaques in RD cells. For the measurement of RNA copy number, the real time quantitative reverse transcription PCR (qRT-PCR) was used. The most potent compound was further evaluated to determine the mode of action of inhibition by time course, virus attachment and entry assays in Vero cells.ResultsSilymarin was shown to exert direct extracellular virucidal effects against EV-A71 at 50% inhibitory concentration (IC50) of 15.2 ± 3.53 μg/mL with SI of 10.53. Similarly, baicalein exhibited direct extracellular virucidal effects against EV-A71 at a higher IC50 value of 30.88 ± 5.50 μg/mL with SI of 13.64. Besides virucidal activity, silymarin was shown to block both viral attachment and entry of EV-A71 to inhibit infection in Vero cells.ConclusionsSilymarin has a stronger inhibition activity against EV-A71 in comparison to baicalein. It could serve as a promising antiviral drug to treat EV-A71 infections.

The Epidemiological and Clinical Characteristics of Hand, Foot, and Mouth Disease in Hangzhou, China, 2016 to 2018.

Hand, foot, and mouth disease (HFMD) is most frequently caused by several serotypes of human enterovirus (EV) including Enterovirus 71 (EV-A71), coxsackievirus A16 (CV-A16), or other types of EV. The aim of this study was to determine the epidemiological characteristics of HFMD and to describe the epidemiologic characteristics of HFMD among severe and mild cases. We collected 4760 HFMD cases in Hangzhou from 2016 to 2018. Specimens from these cases were collected and tested for EV-A71, CV-A16, CV-A6, CV-A10, CV-A2, and CV-A5 by reverse transcriptase polymerase chain reaction. From 2016 to 2018, the prevalence of HFMD was seasonal each year. Among the 4760 probable HFMD cases, 3559 cases were confirmed (74.8%), including 426 cases of EV-A71 infections (8.9%), 249 cases of CV-A16 infections (5.2%), and 2884 cases of other EV infections (60.6%). The percentage of other EV infections was more than 80%, which increased year by year. Random selection of samples for detection of other EV infections in 2017 and 2018, among the 1297 cases, showed there were 835 (64.4%) cases of CV-A6 infections, 177 (13.6%) cases of CV-A10 infections, 100 (7.7%) cases of CV-A2 infections, 40 (3.1%) cases of CV-A5 infections, 3 (0.02 %) cases of mixed infections, and 11.0% untyped EV infections. Preschool children were still the primary population susceptible to HFMD. In severe cases, EV-A71 infection was the main cause. Characterizing the epidemiology and the relationship between severe and common cases of HFMD would provide relevant evidences for the prevention and treatment of HFMD.

MicroRNA 876-5p modulates EV-A71 replication through downregulation of host antiviral factors

BackgroundHuman enterovirus 71 (EV-A71) is a non-enveloped virus that has a single stranded positive sense RNA genome. In a previous study, we showed that miR-876-5p upregulation was observed in the serum of patients with severe EV-A71 infection. Micro-876-5p (miR-876-5p) is a circulating miRNA that can be identified to modulate EV-A71 infections through both in vitro and in vivo studies. However, the regulatory mechanisms that involve miR-876-5p in the EV-A71 infection cycle remain unclear.MethodsWe demonstrated that miR-876-5p facilitated EV-A71 replication and expression by overexpression and knocking-down of miR-876-5p through the transfection of miR-876-5p plasmid and miR-876-5p inhibitor. Although miR-876-5p suppressed CREB5 expression, luciferase reporter assay confirmed this. We also evaluated the role of miR-876-5p in the EV-A71 infection cycle by CREB5 mediated by transfection with an anti-miR-876-5P inhibitor or in combination with an si-CREB5 plasmid.ResultsMicroR-876-5p was upregulated in EV-A71-infected neuroblastoma cells. Overexpression of miR-876-5p or knockdown of cyclic-AMP responsive element binding protein 5 (CREB5) promoted EV-A71 replication. The downregulation of miR-876-5p inhibited the accumulation of viral RNA and the production of viral proteins. Interestingly, CREB5 overexpression also suppressed EV-A71 replication. Our in vitro studies reveal that miR-876-5p directly targets CREB5. Finally, downregulation of CREB5 protein abated the inhibitory effect of anti-miR-876-5p and induced inhibitory effect of EV-A71 replication.ConclusionsOur results suggest that intracellular miR-876-5p promotes EV-A71 replication indirectly by targeting the host CREB5 protein.

Enterovirus infection in the Socialist Republic of Vietnam

Human enterovirus infections comprise a group of infectious diseases caused by viruses of Enterovirus A-D species (genus Enterovirus, family Picornaviridae). Enterovirus infections can vary in clinical manifestations and severity, from asymptomatic infection to serious multisystem diseases. During evolution, enterovirus strains with increased neurovirulence or atypical pathogenicity may emerge exhibiting an epidemic potential. Recently, outbreaks of enterovirus infection with an increased rate of neurological manifestations, a significant percentage of severe cases and lethal outcomes have been observed worldwide, which were associated with enteroviruses EV-A71, EV-D68 etc. The World Health Organization has included EV-A71 and EV-D68 enterovirus infection together with some other dangerous viral diseases considered for inclusion in the List of Blueprint Priority Diseases. In connection with this, global enterovirus surveillance is important for controlling emergence and spread of epidemic enterovirus variants, prediction of establishing epidemic situation, timely conduction of preventive measures and vaccine development. A growing multi-field cooperation between Russia and Vietnam leads to increased two-way population migration, which actualizes scientific and practical collaboration in surveillance and control of infectious disease spread, including enterovirus infection. Currently, epidemiological surveillance of enterovirus infection in Vietnam is based on monitoring hand, foot and mouth disease (HFMD) rate, laboratory diagnostics of enterovirus infection and identification of enterovirus strains, mainly detected in severe patients. In 2001–2016, 34 non-polio virus types were identified in patients with enterovirus infection, largely represented by viruses EV-A71, CVA6, CVA10, and CVA16. Moreover, the peak incidence of enterovirus infection and related mortality rate were associated with the increased activity of EV-A71 virus. In Vietnam, EVA71 enterovirus of genotypes C1, C4, C5 and B5 circulated at different times. Over the last years, a new pandemic genotype virus CVA6 has been dominating as a causative agent of enterovirus infection in Vietnam as well as the majority of other countries. The data on phylogenetic relation between Vietnamese epidemic EV-A71 and CVA6 strains allowed to find that they underwent multiple betweencountry spreads, whereas their subsequent in-country dissemination resulted in 2011–2012 enterovirus outbreak and sustained high-level HFMD morbidity.

Hypoxia and therapeutic treatment of EV-A71 with an immune modulator TLR7 agonist in a new immunocompetent mouse model

BackgroundEnterovirus 71 (EV71 or EV-A71) was first identified in California about half a century ago. In recent years, outbreaks of EV-A71 were prevalent worldwide, including Taiwan, Malaysia, Singapore, Japan, and China. Between 2008 and 2011, China alone reported 1894 deaths associated with EV-A71 infection. In mild cases, EV-A71 can cause herpangina and hand-foot-and-mouth disease (HFMD). However, in severe cases, it could cause neurological disorders, including meningitis and encephalitis. Cardiopulmonary failure is common among hospitalized children with EV-A71 infection. No effective FDA-approved therapeutics against EV-A71 are clinically available.MethodsWe report the establishment of an immunocompetent wild type strain 129 (wt-129) mouse model, which can be cross-species infected with human EV-A71 clinical isolates via an intraperitoneal route.ResultsOne intriguing disease phenotype of this new model is the development of characteristic “White-Jade” patches in the muscle, which lost sporadically the normal pink color of uninfected muscle. Viral VP1 protein and massive leukocyte infiltration were detected in muscles with or without white-jades. We demonstrated further that hypoxia is a general phenomenon associated with white-jades in both immunocompetent and immunodeficient mouse models. Therefore, hypoxia appears to be a feature intrinsic to EV-A71 infection, irrespective of its host’s immunogenetic background. To date, no effective treatment for EV-A71 is available. Here, using this new wt-129 mouse model, we showed that timely treatment with compound R837 (a TLR7 immune modulator) via oral or intraperitoneal routes, rescued the hypoxia, limb paralysis, and death at a high therapeutic efficacy.ConclusionsIn this new immunocompetent mouse 129 model, we observed an unexpected white-jade phenotype and its associated hypoxia. The successful treatment with TLR7 immune modulators via an oral route, provide us a new research direction for EV-A71 basic science and translational research. It remains an open issue whether R837 or its related compounds, will be a promising drug candidate in clinical trials in EV-A71 endemic or epidemic areas in the future.

The Clinical and Epidemiological Study of Children with Hand, Foot, and Mouth Disease in Hunan, China from 2013 to 2017

Hand, foot, and mouth disease (HFMD) is endemic in the Pacific region, especially in mainland China. The case-fatality ratio of HFMD is increasing steadily. Knowledge of the changing epidemiology of HFMD in different regions is necessary for implementing appropriate intervention strategies. In this study, we describe the clinical and epidemiological characteristics of HFMD in Hunan Children’s Hospital between 2013 and 2017. A total of 7203 patients with HFMD were admitted, with complication and mortality rates of 35.62% and 0.78%, respectively. The total number of children with HFMD, proportion of severely ill children, and HFMD mortality rate were the highest in 2014. The number of cases caused by EV-A71 and CV-A16 decreased continuously, while the number of cases caused by ‘other enteroviruses’ increased yearly since 2014, suggesting that other enteric viruses will gradually replace EV-A71 and CV-A16 as the main pathogenic HFMD agents. Furthermore, EV-A71 and mixed infections accounted for the high case fatality rates in children with severe HFMD, among whom EV-A71 infection resulted in the highest complication and mortality rates; the mild form of the disease was dominated by ‘other enteroviruses’. In conclusion, the changing etiological pattern highlights the need to improve pathogen surveillance and vaccine strategies for HFMD control.

Pim1 Impacts Enterovirus A71 Replication and Represents a Potential Target in Antiviral Therapy.

SummaryEnterovirus A71 (EV-A71) infection causes hand-foot-and-mouth disease (HFMD) and fatal neurological diseases, and there are no effective treatments. Host factors play key roles in establishing viral infection and determining the disease progression and outcome of antiviral therapies. In this study, we found that the expression of Pim1 was significantly upregulated in EV-A71 infection. Ectopic expression or silencing of Pim1 promoted or inhibited EV-A71 replication through two distinct mechanisms. Pim1 enhanced viral IRES activity by increasing viral 2A protease-mediated eIF4G cleavage and blocked AUF1, a suppressor of IRES, translocation from the nucleus to cytosol. More importantly, we discovered that Pim1 inhibitors (SGI-1776, AZD-1208, and CX-6258) reduced EV-A71 reproduction. Particularly, CX-6258 remarkably reduced EV-A71 reproduction more than 1,000 times, providing a potential therapeutic agent for EV-A71 treatment.

Global cytokine/chemokine profile identifies potential progression prediction indicators in hand-foot-and-mouth disease patients with Enterovirus A71 infections

ObjectiveNew clinical indicators are urgently needed for predicting the progression and complications of hand-foot-and-mouth disease (HFMD) caused by EV-A71 infections. Materials and methodsSerum specimens from 132 EV-A71 HFMD patients and 73 health children were collected during 2012–2014 in Shenzhen, China. The specific cytokines/chemokines were detected with a 274-human cytokine antibody array, followed by a 38-inflammation cytokine array, and further validated by ELISA. ResultsCytokines varied in different severity of EV-A71 HFMD patients. The ROC curve analysis revealed 5 serum cytokines with high sensitivity and specificity in predicting the disease progression. Eotaxin, IL-8 and IP-10 have showed high AUC values (0.90–0.95) for discrimination between the health controls and the patient group. The three cytokines showed high sensitivity (80–91%) and specificity (88–95%). MMP-8 had a high sensitivity and specificity to predict mild HFMD (100%, 100%). IL-1b and leptin discriminated the severe/critical group from the mild group (79% and 69% in sensitivity, 73% and 63% in specificity). ConclusionsEotaxin, IP-10 and IL-8 could be potential indicators for predicting HFMD progression with EV-A71 infection. MMP-8 is a specific indicator for mild infection, while IL-1b and leptin display potential for predicting the severity and criticality.

Hsp27 Responds to and Facilitates Enterovirus A71 Replication by Enhancing Viral Internal Ribosome Entry Site-Mediated Translation.

Enterovirus 71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease (HFMD) and fatal neurological diseases, and no effective treatment is available. Characterization of key host factors is important for understanding its pathogenesis and developing antiviral drugs. Here we report that Hsp27 is one of the most upregulated proteins in response to EV-A71 infection, as revealed by two-dimensional gel electrophoresis-based proteomics studies. Depletion of Hsp27 by small interfering RNA or CRISPR/Cas9-mediated knockout significantly inhibited viral replication, protein expression, and reproduction, while restoration of Hsp27 restored such virus activities. Furthermore, we show that Hsp27 plays a crucial role in regulating viral internal ribosome entry site (IRES) activities by two different mechanisms. Hsp27 markedly promoted 2Apro-mediated eukaryotic initiation factor 4G cleavage, an important process for selecting and initiating IRES-mediated translation. hnRNP A1 is a key IRES trans-acting factor (ITAF) for enhancing IRES-mediated translation. Surprisingly, knockout of Hsp27 differentially blocked hnRNP A1 but not FBP1 translocation from the nucleus to the cytoplasm and therefore abolished the hnRNP A1 interaction with IRES. Most importantly, the Hsp27 inhibitor 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone (TDP), a compound isolated from a traditional Chinese herb, significantly protected against cytopathic effects and inhibited EV-A71 infection. Collectively, our results demonstrate new functions of Hsp27 in facilitating virus infection and provide novel options for combating EV-A71 infection by targeting Hsp27.IMPORTANCE Outbreaks of infections with EV-A71, which causes hand, foot, and mouth disease, severe neurological disorders, and even death, have been repeatedly reported worldwide in recent decades and are a great public health problem for which no approved treatments are available. We show that Hsp27, a heat shock protein, supports EV-A71 infection in two distinct ways to promote viral IRES-dependent translation. A small-molecule Hsp27 inhibitor isolated from a traditional Chinese medicinal herb effectively reduces virus yields. Together, our findings demonstrate that Hsp27 plays an important role in EV-A71 infection and may serve as an antiviral target.

Elucidating the Host Interactome of EV-A71 2C Reveals Viral Dependency Factors.

Viral protein 2C plays a critical role in EV-A71 replication. The discovery of 2C binding proteins will likely provide potential targets to treat EV-A71 infection. Here, we provide a global proteomic analysis of the human proteins that interact with the EV-A71 2C protein. TRIM4, exportin2, and ARFGAP1 were validated as 2C binding partners. Further functional studies revealed that TRIM4, exportin2, and ARFGAP1 were novel host dependency factors for EV-A71. Moreover, enteroviruses’ 2C family proteins interacted with exportin2 and ARFGAP1. In conclusion, our study provides a cellular interactome of the EV-A71 2C and identifies the proviral roles of TRIM4, exportin2, and ARFGAP1 in EV-A71 infection.

Molecular epidemiology of an enterovirus A71 outbreak associated with severe neurological disease, Spain, 2016.

IntroductionEnterovirus A71 (EV-A71) is an emerging pathogen that causes a wide range of disorders including severe neurological manifestations. In the past 20 years, this virus has been associated with large outbreaks of hand, foot and mouth disease with neurological complications in the Asia-Pacific region, while in Europe mainly sporadic cases have been reported. In spring 2016, however, an EV-A71 outbreak associated with severe neurological cases was reported in Catalonia and spread further to other Spanish regions.AimOur objective was to investigate the epidemiology and clinical characteristics of the outbreak.MethodsWe carried out a retrospective study which included 233 EV-A71-positive samples collected during 2016 from hospitalised patients. We analysed the clinical manifestations associated with EV-A71 infections and performed phylogenetic analyses of the 3’-VP1 and 3Dpol regions from all Spanish strains and a set of EV-A71 from other countries.ResultsMost EV-A71 infections were reported in children (mean age: 2.6 years) and the highest incidence was between May and July 2016 (83%). Most isolates (218/233) were classified as subgenogroup C1 and 217 of them were grouped in one cluster phylogenetically related to a new recombinant variant strain associated with severe neurological diseases in Germany and France in 2015 and 2016. Moreover, we found a clear association of EV-A71-C1 infection with severe neurological disorders, brainstem encephalitis being the most commonly reported.ConclusionAn emerging recombinant variant of EV-A71-C1 was responsible for the large outbreak in 2016 in Spain that was associated with many severe neurological cases.