Enterovirus A71 Infection Research Articles

Inhibition of Enterovirus A71 by a Novel 2-Phenyl-Benzimidazole Derivative.

Enterovirus A71 (EV-A71) infection has emerged as a significant public health concern at the global level. Epidemic events of EV-A71 have been reported worldwide, and this succession of outbreaks has heightened concern that EV-A71 may become a public health threat. In recent years, widespread A71 enterovirus also occurred in European countries. EV-A71 infection causes hand-foot-mouth disease (HFMD), herpangina, and fever. However, it can sometimes induce a variety of neurological complications, including encephalitis, aseptic meningitis, pulmonary edema, and acute flaccid paralysis. We identified new benzimidazole derivatives and described theirin vitrocytotoxicity and broad-spectrum anti-enterovirus activity. Among them, derivative 2b resulted in interesting activity against EV-A71, and therefore it was selected for further investigations. Compound 2b proved to be able to protect cell monolayers from EV-A71-induced cytopathogenicity, with an EC50 of 3 µM. Moreover, Vero-76 cells resulted in being significantly protected from necrosis and apoptosis when treated with 2b at 20 and 80 µM. Compound 2b reduced viral adsorption to Vero-76 cells, and when evaluated in a time-of-addition assay, the derivative had the highest effect when added during the infection period. Moreover, derivative 2b reduced viral penetration into host cells. Besides, 2b did not affect intestinal monolayers permeability, showing no toxic effects. A detailed insight into the efficacy of compound 2b against EV-A71 showed a dose-dependent reduction in the viral titer, also at low concentrations. Mechanism of action investigations suggested that our derivative can inhibit viral endocytosis by reducing viral attachment to and penetration into host cells. Pharmacokinetic and toxicity predictions validated compound 2b as a good candidate for furtherin vivoassays.

Engineering a novel IgG-like bispecific antibody against enterovirus A71

Frequent outbreaks of enterovirus A71 (EVA71) occur in the Asia-Pacific area, and these are closely associated with severe neurological symptoms in young children. No effective antiviral therapy is currently available for the treatment of EVA71 infection. The development of monoclonal antibodies (mAbs) has demonstrated promise as a novel therapy for the prevention and treatment of infectious diseases. Several medical conditions have been treated using bispecific or multi-specific antibodies that recognize two or more distinct epitopes simultaneously. However, bispecific or multi-specific antibodies often encounter protein expression and product stability problems. In this study, we developed an IgG-like bispecific antibody (E18-F1) comprising two anti-EVA71 antibodies: E18 mAb and llama-derived F1 single-domain antibody. E18-F1 was demonstrated to exhibit superior binding affinity and antiviral activity compared with E18 or F1. Additionally, E18-F1 not only improved survival rate, but also reduced clinical signs in human SCARB2 receptor (hSCARB2) transgenic mice challenged with a lethal dose of EVA71. Altogether, our results reveal that E18-F1 is a simple format bispecific antibody with promising antiviral activity for EVA71.

A longitudinal cohort study of enterovirus A71 infections in young children in Taiwan

Background: Enterovirus A71 (EV-A71) is causing cyclical epidemics of fatal hand-foot-mouth disease (HFMD) in Taiwan and other Asian countries. Evidence of its incidence rates and risk factors based on longitudinal cohort studies is scarce. This study aims to investigate age-specific and cumulative incidence rates and risk factors of EV71 infections from 2006 to 2012 in northern Taiwan. Methods and materials: A total of 759 neonates born between 2006 and 2009 were recruited. Participants’ sera were collected at their 6, 12, 24, 36, 48, 60, and 72 months of age to conduct serum neutralization test. In addition, throat swabs of the participants who presented symptoms of HFMD or herpangina were collected for virus isolation and molecular tests to detect EV71 infections. Asymptomatic primary infections and reinfections were identified based on >= 4-fold rise of serum neutralizing antibody titers in consecutive sera. We calculated age-specific and cumulative incidence rates, and fitted a Cox proportional hazards model to identify risk factors of EV71 infections. Results: Among the 576 participants being followed, 127 persons (22.0%) were confirmed as with EV71 infections (70 symptomatic, 57 asymptomatic cases). Of the 127 cases, 22 (17.3%) occurred their second (n = 20) or third (n = 2) reinfections (all asymptomatic) after the primary infection. Phylogenetic analysis revealed that two distinguishable genotype B5 viruses were introduced from southern Asia to cause large-scale epidemics in Taiwan in 2008 and 2012, respectively. Age-specific incidence increased from 2.03 per 100 person-years during 0–6 months of age to 19.33 during 49–60 months of age, and reduced to 9.57 during 61–72 months of age. The cumulative incidence till 72 months of age was 31.78%. A higher number of co-residing children under 5 years old was associated with higher risk of EV71 infections (hazard ratio = 1.28, 95% CI: 1.03–1.59). Conclusion: Our results indicated incidence rates increased with age and peaked at 5 years-old, cumulative incidence rate was nearly 32% by 6 years of age, and higher number of co-residing young children was associated with higher risk of EV71 infections. This longitudinal cohort study contributes to precisely estimate incidence rates of EV71 infections via a long-term follow-up, and provides insights into clinical trial designs of EV71 vaccines.

The transfer and decay of maternal antibodies against enterovirus A71, and dynamics of antibodies due to later natural infections in Chinese infants: a longitudinal, paired mother–neonate cohort study

Since 1997, epidemics of hand, foot, and mouth disease associated with enterovirus A71 (EV-A71) have affected children younger than 5 years in the Asia-Pacific region, including mainland China. EV-A71 vaccines have been licensed for use in children aged 6-71 months in China, but not for infants younger than 6 months. We aimed to assess the dynamics of maternal EV-A71 antibodies to inform choice of potential vaccination strategies to protect infants younger than 6 months, because they have a substantial burden of disease. We did a longitudinal cohort study with mother-neonate pairs in local hospitals in southern China during 2013-18. We collected cord blood from neonates and venous blood from mothers at delivery. We followed up and collected blood samples from the children at ages 2, 4, 6, 12, 24, and 36 months and tested for the presence of neutralising antibodies against EV-A71 with virus neutralisation assays. Seropositivity, or protective titre, was defined as a neutralisation antibody titre of 16 or higher. We estimated the seroprevalence, geometric mean titre (GMT), and transfer ratio of maternal antibodies. We used a binomial distribution to derive the 95% CIs of seroprevalence. Seropositivity between mothers and neonates was compared by use of an agreement (κ), while GMTs were compared by use of paired Student's t tests. Between Sept 20, 2013, and Oct 14, 2015, 1054 mothers with 1066 neonates were enrolled. The EV-A71 GMT was similar among pairs of neonates (22·7, 95% CI 20·8-24·9) and mothers (22·1, 95% CI 20·2-24·1; p=0·20). The mean transfer ratio of maternal antibodies was 1·03 (95% CI 0·98-1·08). Although 705 (66%) of 1066 neonates acquired protective concentrations of EV-A71 antibodies from mothers, these declined rapidly, with a half-life of 42 days (95% CI 40-44). The time to loss of protective immunity was extended to 5 months in neonates with mothers who had titres of 128 or higher. By age 30 months, 28% of children had become seropositive because of natural infection. EV-A71 maternal antibodies were efficiently transferred to neonates, but declined quickly to below the protective threshold, particularly among those whose mothers had low antibody titres. Our findings suggest that maternal vaccination could be explored to provide neonatal protection against EV-A71 through maternal antibodies. Catch-up vaccination between ages 6 months to 5 years could provide protection to the approximately 30-90% of children that have not had natural EV-A71 infection by that age. National Science Fund for Distinguished Young Scholars, National Natural Science Foundation of China.

A Polysaccharide Purified From Ganoderma lucidum Acts as a Potent Mucosal Adjuvant That Promotes Protective Immunity Against the Lethal Challenge With Enterovirus A71.

Enterovirus A71 (EV-A71), the pathogen responsible for the seasonal hand-foot-and-mouth epidemics, can cause significant mortality in infants and young children. The vaccine against EV-A71 could potentially prevent virus-induced neurological complications and mortalities occurring due to the high risk of poliomyelitis-like paralysis and fatal encephalitis. It is known that polysaccharide purified from Ganoderma lucidum (PS-G) can effectively modulate immune function. Here, we used PS-G as an adjuvant with the EV-A71 mucosal vaccine and studied its effects. Our data showed that PS-G-adjuvanted EV-A71 generated significantly better IgA and IgG in the serum, saliva, nasal wash, bronchoalveolar lavage fluid (BALF), and feces. More importantly, these antibodies could neutralize the infectivity of EV-A71 (C2 genotype) and cross-neutralize the B4, B5, and C4 genotypes of EV-A71. In addition, more EV-A71-specific IgA- and IgG- secreting cells were observed with the used of a combination of EV-A71 and PS-G. Furthermore, T-cell proliferative responses and IFN-γ and IL-17 secretions levels were notably increased in splenocytes when the EV-A71 vaccine contained PS-G. We also found that levels of IFN-γ and IL-17 released in Peyer’s patch cells were significantly increased in EV-A71, after it was combined with PS-G. We further demonstrated that both CD4+ and CD8+ T cells were able to generate IFN-γ and IL-17 in the spleen. An easy-accessed model of hybrid hSCARB2+/+/stat-1–/– mice was used for EV-A71 infection and pathogenesis. We infected the mouse model with EV−A71, which was premixed with mouse sera immunized with the EV-A71 vaccine with or without PS-G. Indeed, in the EV-A71 + PS-G group, the levels of VP1-specific RNA sequences in the brain, spinal cord, and muscle decreased significantly. Finally, hSCARB2-Tg mice immunized via the intranasal route with the PS-G-adjuvanted EV-A71 vaccine resisted a subsequent lethal oral EV-A71 challenge. Taken together, these results demonstrated that PS-G could potentially be used as an adjuvant for the EV-A71 mucosal vaccine.

Autophagy is induced and supports virus replication in Enterovirus A71-infected human primary neuronal cells

Enterovirus A71 (EV-A71), which belongs to the family Picornaviridae, can invade the central nervous system (CNS) and cause severe CNS complications or death. The EV-A71 antigen has been detected in the neurons in the brains of humans who died from EV-A71 infection. However, the effect of EV-A71 infection on human neuronal cells remains poorly understood. Human neural stem cells (NSCs) and IMR-32 neuroblastoma cells were differentiated into neuronal cells for this study. Although the neuronal cells were permissive to EV-A71 infection, EV-A71 infection did not induce an obvious cytopathic effect on the neuronal cells. EV-A71 infection did not induce apoptosis in neuronal cells. However, autophagy and autophagic flux were induced in EV-A71-infected neuronal cells. The production of autophagosomes was shown to be important for EV-A71 viral RNA (vRNA) replication in neuronal cells.

Evaluation of the diagnostic performance and its associated factors of a commercial anti-EV-A71 IgM-capture ELISA kit in hospitalized children with clinical diagnostic HFMD.

Enterovirus A71 (EV-A71) is the main pathogen of severe hand, foot, and mouth disease (HFMD). Commercial enzyme-linked immunosorbent assays (ELISAs) are widely used in Chinese hospitals for the rapid diagnosis of acute EV-A71 infections. We present an evaluation of the diagnostic performance of a commercial anti-EV-A71 IgM-capture ELISA kit. A prospective, hospital-based HFMD cohort was established in Henan Children's Hospital (February 2017 - February 2018). Stool and blood specimens were collected from 1413 participants for diagnosing EVA71 by quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and anti-EV-A71 ELISA. Detection yields of EV-A71 IgM increased from 6.5 % (95 % CI:3.3 %-11.4 %) at 0∼24 h, to 42 % (95 % CI:28.3 %-57.8) at 120∼144 h from onset to sampling, and stabilized at ∼40 % after 144 h. With increased time from onset to sampling, the sensitivity of the commercial ELISA increased from 0.54 (95 % CI:0.25-0.81) to 0.74 (95 % CI:0.43-0.66), while specificity decreased from 0.97 (95 % CI:0.93-0.99) to 0.80 (95 % CI:0.69-0.89), and PPV decreased from 0.96 (95 % CI:0.92-0.99) to 0.84 (95 % CI:0.73-0.92). Multivariate analysis found age, EV-A71 vaccination, previous HFMD/Herpangina infection, disease severity, infection during peak EV-A71 season, and sampling time after symptom onset were significantly associated with the diagnostic performance of this anti-EV-A71 IgM-capture ELISA. Achieving satisfactory specificity and sensitivity scores, this commercial anti-EV-A71 IgM-capture ELISA kit is suitable for clinical EV-A71 diagnosis, particularly in resource-poor areas. However, clinicians should interpret results in the context of patient history and epidemiological setting.

Metabolic Profiling Reveals Significant Perturbations of Intracellular Glucose Homeostasis in Enterovirus-Infected Cells

Enterovirus A71 (EV-A71) is a common cause of hand, foot, and mouth disease. Severe EV-A71 infections may be associated with life-threatening neurological complications. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Metabolites are known to play critical roles in multiple stages of the replication cycles of viruses. The metabolic reprogramming induced by viral infections is essential for optimal virus replication and may be potential antiviral targets. In this study, we applied targeted metabolomics profiling to investigate the metabolic changes of induced pluripotent human stem cell (iPSC)-derived neural progenitor cells (NPCs) upon EV-A71 infection. A targeted quantitation of polar metabolites identified 14 candidates with altered expression profiles. A pathway enrichment analysis pinpointed glucose metabolic pathways as being highly perturbed upon EV-A71 infection. Gene silencing of one of the key enzymes of glycolysis, 6-phosphofructo-2-kinase (PFKFB3), significantly suppressed EV-A71 replication in vitro. Collectively, we demonstrated the feasibility to manipulate EV-A71-triggered host metabolic reprogramming as a potential anti-EV-A71 strategy.

TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71.

Enterovirus A71 (EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease (HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3C between the residues 331Q and 332S, while mutated TDP-43 (Q331A) was not cleaved. In addition, mutated 3C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2A rather than 3C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71infection.

The Heat Shock Protein 70 Family of Chaperones Regulates All Phases of the Enterovirus A71 Life Cycle.

Enterovirus A71 (EV-A71) is one of the major etiologic agents causing hand, foot, and mouth disease (HFMD) in children and occasionally causes severe neurological diseases or even death. EV-A71 replicates rapidly in host cells. For a successful infection, viruses produce large quantities of viral proteins in a short period, which requires cellular chaperone proteins for viral protein folding and viral particle assembly. In this study, we explored the roles of the heat shock protein 70 (HSP70) chaperone subnetwork in the EV-A71 life cycle. Our results revealed that EV-A71 exploits multiple HSP70s at each step of the viral life cycle, i.e., viral entry, translation, replication, assembly and release, and that each HSP70 typically functions in several stages of the life cycle. For example, the HSP70 isoforms HSPA1, HSPA8, and HSPA9 are required for viral entry and the translational steps of the infection. HSPA8 and HSPA9 may facilitate folding and stabilize viral proteins 3D and 2C, respectively, thus contributing to the formation of a replication complex. HSPA8 and HSPA9 also promote viral particle assembly, whereas HSPA1 and HSPA8 are involved in viral particle release. Because of the importance of various HSP70s at distinct steps of the viral life cycle, an allosteric inhibitor, JG40, which targets all HSP70s, significantly blocks EV-A71 infection. JG40 also blocks the replication of several other enteroviruses, such as coxsackievirus (CV) A16, CVB1, CVB3, and echovirus 11. Thus, targeting HSP70s may be a means of providing broad-spectrum antiviral therapy.

A Mouse Model for Infection with Enterovirus A71 in Small Extracellular Vesicles.

Enterovirus A71 (EV-A71) is the major pathogen of hand, foot, and mouth disease (HFMD); in some severe cases, it could develop into central nervous system (CNS) disease such as aseptic meningitis, encephalitis, and neurogenic pulmonary edema in children under 5 years. The EV-A71 pathogenesis which is involved with the CNS is unclear due to the lack of a simple and reliable mouse model thus far. Most clinical EV-A71 isolates could not effectively infect the neonatal mouse, which used to be an EV-A71 infection model. The small extracellular vesicles (sEVs) released from clinical EV-A71 isolate-infected cells were infectious in cell lines and could cause a high viral replication in mice. Neonatal ICR mice were injected intraperitoneally with these infectious sEVs and showed more weight loss and higher mortality than those mice injected with the clinical EV-A71 isolate. By using these sEVs, we provided a simple and effective method by which we can generate a stable and valuable animal model for the studies of EV-A71 pathogenesis and therapy.IMPORTANCE EV-A71 was supposed to infect the CNS through the neural pathway and the circulation of the blood in previous studies. Reverse axon transport had been confirmed as an important pathway for EV-A71 to infect the CNS; however, it is still unknown how EV-A71 infects the CNS through the circulation of the blood. Combined with the infectivity of sEVs secreted from EV-A71-infected cells and the characteristic that sEVs could cross the blood-brain barrier, we considered that sEVs may play a vital role in EV-A71 pathogenesis of the CNS.

Identification of dibucaine derivatives as novel potent enterovirus 2C helicase inhibitors: In vitro, in vivo, and combination therapy study

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC50 = 1.238 μM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.

Rac1-dependent endocytosis and Rab5-dependent intracellular trafficking are required by Enterovirus A71 and Coxsackievirus A10 to establish infections

Enterovirus A71 (EVA71) and Coxsackievirus A10 (CVA10) are representative types of Enterovirus A. Dependent on the host cell types, the EVA71 entry may utilize clathrin-, caveola-, and endophilin-A2-mediated endocytosis. However, the cell-entry and intracellular trafficking pathways of CVA10, using KREMEN1 as its receptor, are unclear. Here, we tested the relevant mechanisms through RNA interference (RNAi) and chemical inhibitors. We found that endocytosis of EVA71 and CVA10 in rhabdomyosarcoma (RD) cells engaged multiple pathways, and both viruses required Rac1. Interestingly, while CDC42 and Pak1 participated in EVA71 infection, PI3K played a role in CVA10 infection. The functions of Rab proteins in intracellular trafficking of CVA10 and EVA71 were examined by RNAi. Knockdown of Rab5 and Rab21 significantly reduced CVA10 infectivity, while knockdown of Rab5, Rab7 and Rab9 reduced EVA71 infectivity. Confocal microscopy confirmed the colocalization of CVA10 virions with Rab5 or Rab21, and colocalization of EVA71 virions with Rab5 or Rab7. Additionally, we observed that both CVA10 and EVA71 infections were inhibited by endosome acidification inhibitors, bafilomycin-A1 and NH4Cl. Together, our findings comparatively illustrate the entry and intracellular trafficking processes of representative Enterovirus A types and revealed novel enterovirus intervention targets.

Drug repurposing of pyrimidine analogs as potent antiviral compounds against human enterovirus A71 infection with potential clinical applications

Enterovirus A71 (EV-A71) is one of the aetiological agents for the hand, foot and mouth disease (HFMD) in young children and a potential cause of neurological complications in afflicted patients. Since its discovery in 1969, there remains no approved antiviral for EV-A71 and other HFMD-causing enteroviruses. We set out to address the lack of therapeutics against EV-A71 by screening an FDA-approved drug library and found an enrichment of hits including pyrimidine antimetabolite, gemcitabine which showed 90.2% of inhibition on EV-A71 infection. Gemcitabine and other nucleoside analogs, LY2334737 and sofosbuvir inhibition of EV-A71 infection were disclosed using molecular and proteomic quantification, and in vitro and in vivo efficacy evaluation. Gemcitabine displayed a significant reduction of infectious EV-A71 titres by 2.5 logs PFU/mL and was shown to target the early stage of EV-A71 viral RNA and viral protein synthesis process especially via inhibition of the RNA dependent RNA polymerase. In addition, the drug combination study of gemcitabine’s synergistic effects with interferon-β at 1:1 and 1:2 ratio enhanced inhibition against EV-A71 replication. Since gemcitabine is known to metabolize rapidly in vivo, other nucleoside analogs, LY2334737 and sofosbuvir conferred protection in mice against lethal EV-A71 challenge by potentially reducing the death rate, viral titers as well on virus-induced pathology in the limb muscle tissue of mice. Additionally, we found that gemcitabine is competent to inhibit other positive-sense RNA viruses of the Flaviviridae and Togaviridae family. Overall, these drugs provide new insights into targeting viral factors as a broad-spectrum antiviral strategy with potential therapeutic value for future development and are worthy of potential clinical application.

Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71.

In the past few decades, enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region, resulting in serious sequelae in infected young children. No preventive or therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single-domain antibody (sdAb), F1, isolated from an immunized llama, could alleviate EVA71 infection both in vitro and in vivo We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide enzyme-linked immunosorbent assays (ELISAs). Because of the virion's icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two single-domain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens could be further increased by valency effects. We showed that the tetravalent construct F1×F1-hFc, containing two sdAb-in-tandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half dose of the F1×F1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.

Involvement of VCP/UFD1/Nucleolin in the viral entry of Enterovirus A species

Valosin-containing protein (VCP) plays roles in various cellular activities. Recently, Enterovirus A71 (EVA71) infection was found to hijack the VCP protein. However, the mechanism by which VCP participates in the EVA71 life cycle remains unclear. Using chemical inhibitor, RNA interference and dominant negative mutant, we confirmed that the VCP and its ATPase activity were critical for EVA71 infection. To identify the factors downstream of VCP in enterovirus infection, 31 known VCP-cofactors were screened in the siRNA knockdown experiments. The results showed that UFD1 (ubiquitin recognition factor in ER associated degradation 1), but not NPL4 (NPL4 homolog, ubiquitin recognition factor), played critical roles in infections by EVA71. UFD1 knockdown suppressed the activity of EVA71 pseudovirus (causing single round infection) while it did not affect the viral replication in replicon RNA transfection assays. In addition, knockdown of VCP and UFD1 reduced viral infections by multiple human Enterovirus A serotypes. Mechanistically, we found that knockdown of UFD1 significantly decreased the binding and the subsequent entry of EVA71 to host cells through modulating the levels of nucleolin protein, a coreceptor of EVA71. Together, these data reveal novel roles of VCP and its cofactor UFD1 in the virus entry by EVA71.

Interaction between PHB2 and Enterovirus A71 VP1 Induces Autophagy and Affects EV-A71 Infection.

Enterovirus A71 (EV-A71) is a major pathogen that causes severe and fatal cases of hand-foot-and-mouth disease (HFMD). HFMD caused by EV-A71 seriously endangers children’s health. Although autophagy is an important antiviral defense mechanism, some viruses have evolved strategies to utilize autophagy to promote self-replication. EV-A71 can utilize autophagy vesicles as replication scaffolds, indicating that EV-A71 infection is closely related to its autophagy induction mechanism. VP1, a structural protein of EV-A71, has been reported to induce autophagy, but the underlying mechanism is still unclear. In this study, we found that the C-terminus (aa 251–297) of VP1 induces autophagy. Mass spectrometry analysis suggested that prohibitin 2 (PHB2) interacts with the C-terminus of the EV-A71 VP1 protein, and this was further verified by coimmunoprecipitation assays. After PHB2 knockdown, EV-A71 replication, viral particle release, and viral protein synthesis were reduced, and autophagy was inhibited. The results suggest that PHB2 interaction with VP1 is essential for induction of autophagy and the infectivity of EV-A71. Furthermore, we confirmed that EV-A71 induced complete autophagy that required autolysosomal acidification, thus affecting EV-A71 infection. In summary, this study revealed that the host protein PHB2 is involved in an autophagy mechanism during EV-A71 infection.

Mechanism for the lethal effect of enterovirus A71 intracerebral injection in neonatal mice

Enterovirus A71 (EV-A71) infection is primarily responsible for fatal hand, foot, and mouth disease (HFMD) cases. Infants and younger children are more likely to suffer central nervous system damage as a result of EV-A71 infection, but this virus mostly does not affect older children and adults. This study investigated the possible mechanism underlying the age-dependent lethal effect of EV-A71 infection by comparing neonatal and adult mouse models of EV-A71 infection. Although viral proliferation is absent in both neonatal and adult mice, we observed that EV-A71, as a stimulus for astrocytes, elevates the levels of cytokines and monoamine neurotransmitters in neonatal mice. Then, we selected IL-6 and adrenaline as targets in a pharmacological approach to further validate the roles of these factors in mediating the mortality of neonatal mice after EV-A71 infection. Intracerebral injection of IL-6 and adrenaline enhanced the severity of EV-A71 infection, while treatment with an anti-IL-6-neutralizing antibody or the adrenergic-antagonist phenoxybenzamine reversed the lethal effect of EV-A71 in neonatal mice. These results suggest that the central nervous system (CNS) damage in neonatal cases of EV-A71 infection might be caused by an activated fetal cerebral immune response to the virus, including the disruption of brainstem function through increased levels of cytokines and neurotransmitters, rather than the typical cytopathic effect (CPE) of viral infection.

Sodium Copper Chlorophyllin Is Highly Effective against Enterovirus (EV) A71 Infection by Blocking Its Entry into the Host Cell.

Human enteroviruses (HEVs) pose an ongoing threat to global public health. Particularly, enterovirus-A71 (EV-A71), the main pathogen causing hand-foot-and-mouth disease (HFMD), has caused ongoing outbreaks globally in recent years associated with severe neurological manifestations and several deaths. Currently, no effective antivirals are available for the prevention or treatment of EV-A71 infection. In this study, we found that sodium copper chlorophyllin (CHL), a health food additive and an over-the-counter anticancer medicine or treatment to reduce the odor of urine or feces, exhibited potent inhibitory activity against infection by divergent EV-A71 and coxsackievirus-A16 (CV-A16) isolates at a low micromolar concentration with excellent safety. The antiviral activity of each was confirmed by colorimetric viral infection and qRT-PCR assays. A series of mechanistic studies showed that CHL did not target the host cell but blocked the entry of EV-A71 and CV-A16 into the host cell at the postattachment stage. In the mouse model, CHL could significantly reduce the viral titer in the lungs and muscles. Since CHL has been used in clinics for many years with excellent safety, it has the potential to be further developed into a prophylactic or therapeutic to prevent or treat HFMD caused by EV-A71 or CV-A16 infection.

Transcriptome sequencing analysis of SH-SY5Y cells infected with EV71 reveals the potential neuropathic mechanisms

Enterovirus A71 (EV71) remains the most common causative agent of hand, foot, and mouth disease (HFMD), and the neurological complications induced by EV71 are usually the leading cause of death in children with HFMD. However, the mechanism of nervous system changes caused by EV71 infection is still unclear. Therefore, in the current study, EV71 was inoculated into the human neuroblastoma cell line SH-SY5Y and subsequent transcriptome sequencing was used to examine the alterations of the transcriptome in infected SH-SY5Y cells. It is expected to determine the underlying mechanism of neurological diseases in response to EV71 infection. As a result, a total of 82,406,974, 112,410,808 and 87,780,371 clean reads were found in the control, EV71-12 h and EV71-24 h groups, respectively. Moreover, 160 and 745 differentially expressed genes were identified in the EV71-12 h and EV71-24 h groups, respectively, as compared to the control group. Next, to further explore the pathogenic mechanism triggered by EV71 infection, we mainly focused on the common differentially expressed genes at different time points of EV71 infection. And it was discovered that there were 95 common differentially expressed genes, which were used to conduct GO and pathway analysis. GO enrichment analysis demarcated related biological processes, molecular functions and cellular components, and KEGG pathway analysis enabled annotations of metabolic pathways and revealed interactions among the significantly enriched pathways. The results showed that the enriched GO term “Nervous system development” and enriched pathway “CCKR signaling map” might be important contributors to EV71-induced neuropathological mechanisms. In addition, we also screened 10 up- and down-regulated non-protein coding genes with significantly different expression in our transcriptome profiling, which suggested that these abnormally regulated non-protein-encoding genes might also play important roles in the pathogenesis of EV71 infection. Eventually, RT-qPCR technology was adopted to validate the transcriptome sequencing data and the experiment demonstrated that the RT-qPCR and transcriptome sequencing results were basically consistent. In summary, this is the first transcriptome analysis of SH-SY5Y cells in response to EV71 infection and provides valuable cues for further exploring the mechanism of nervous system changes caused by EV71 infection.