Metabolomics combined with network pharmacology reveals a role for astragaloside IV in inhibiting enterovirus 71 replication via PI3K-AKT signaling.

BACKGROUND: Astragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms. METHODS: The GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID50), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman's algorithm. RESULTS: This study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis. CONCLUSION: This study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication.

Correlations of PSGL-1 VNTR polymorphism with the susceptibility to severe HFMD associated with EV-71 and the immune status after infection.

Enterovirus 71 (EV-71) has strong neurotropism, and it is the main pathogen causing severe hand, foot, and mouth disease (HFMD). In clinical observations, significant differences were observed in the severity and prognosis of HFMD among children who were also infected with EV-71. Genetic differences among individuals could be one of the important causes of differences in susceptibility to EV-71-induced HFMD. As P-selectin glycoprotein ligand-1 (PSGL-1) is an important receptor of EV-71, the correlation between single-nucleotide polymorphisms (SNPs) in PSGL-1 and the susceptibility to severe HFMD following EV-71 infection is worth studying. Given the role of PSGL-1 in immunity, the correlations between PSGL-1 SNPs and the immune status after EV-71 infection are also worth studying. Meanwhile, PSGL-1 variable number of tandem repeats (VNTR) represents a research hotspot in cardiovascular and cerebrovascular diseases, but PSGL-1 VNTR polymorphism has not been investigated in HFMD caused by EV-71 infection. In this study, specific gene fragments were amplified by polymerase chain reaction, and PSGL-1 VNTR sequences were genotyped using an automatic nucleic acid analyzer. The correlations of PSGL-1 VNTR polymorphism with the susceptibility to EV-71-associated severe HFMD and the post-infection immune status were analyzed. The PSGL-1 VNTR A allele was identified as a susceptible SNP for severe HFMD. The risk of severe HFMD was higher for AA + AB genotype carriers than for BB genotype carriers. The counts of peripheral blood lymphocyte subsets were lower in AA + AB genotype carries than in BB genotype carries. In conclusion, PSGL-1 VNTR polymorphism is associated with the susceptibility to EV-71-induced severe HFMD and the immune status after infection. PSGL-1 VNTR might play a certain role in the pathogenesis of severe cases.

Isolation and identification of active components from Grifola frondosa and its anti-EV71 virus effect.

BACKGROUND: It is reported that anti-enterovirus 71 (EV71) drugs have some side effects on human health. Notably, fungi plays a crucial role in promoting human health and anti-virus. Grifola frondosa is a type of large medicinal and edible fungi, rich in active substances. The present study aimed to investigate the anti-EV71 effect of G. frondosa and the potential active substances. RESULTS: In the present study, the water extract of G. frondosa was subjected to ethanol precipitation to obtain the water-extracted supernatant of G. frondosa (GFWS) and water-extracted precipitation of G. frondosa. Their inhibitory effects on EV71 virus were studied based on a cell model. The results showed that GFWS had stronger security and anti-EV71 effects. In addition, the chemical constituents of GFWS were identified by ultra-high performance liquid chromatography-tandem mass spectrometry, which were selected for further separation and purification. Three compounds, N-butylaniline, succinic acid and l-tryptophan, were isolated from GFWS by NMR spectroscopy. It is noteworthy that N-butylaniline and l-tryptophan were isolated and identified from the G. frondosa fruiting bodies for the first time. Our study found that l-tryptophan has anti-EV71 virus activity, which reduced EV71-induced apoptosis and significantly inhibited the replication process after virus adsorption. Furthermore, it could also bind to capsid protein VP1 to prevent the virus from attaching to the cells. CONCLUSION: l-tryptophan was an inhibitor of the EV71 virus, which could be used in infant nutrition and possibly provide a new drug to treat hand, foot and mouth disease. © 2024 Society of Chemical Industry.

SLC35B2 Acts in a Dual Role in the Host Sulfation Required for EV71 Infection.

As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young children. Understanding the interaction between host factors and EV71 will play a vital role in developing antivirals and optimizing vaccines. Here, we performed a genome-wide CRISPR-Cas9 knockout screen and revealed that scavenger receptor class B member 2 (SCARB2), solute carrier family 35 member B2 (SLC35B2), and beta-1,3-glucuronyltransferase 3 (B3GAT3) are essential in facilitating EV71 replication. Subsequently, the exploration of molecular mechanisms suggested that the knockout of SLC35B2 or B3GAT3, not SCARB2, led to a remarkable decrease in the binding of EV71 to cells and internalization into cells. Furthermore, we found that the infection efficiency for EV71 was positively correlated with the level of host cell sulfation, not simply with the amount of heparan sulfate, suggesting that an unidentified sulfated protein(s) must contribute to EV71 infection. In support of this idea, we screened possible sulfated proteins among the proteinous receptors for EV71 and confirmed that SCARB2 could uniquely interact with both tyrosyl protein sulfotransferases in humans. We then performed mass spectrometric analysis of SCARB2, identifying five sites with tyrosine sulfation. The function verification test indicated that there were more than five tyrosine-sulfated sites on SCARB2. Finally, we constructed a model for EV71 entry in which both heparan sulfate and SCARB2 are regulated by SLC35B2 and act cooperatively to support viral binding, internalization, and uncoating. Taken together, this is the first time that we performed the pooled CRISPR-Cas9 genetic screening to investigate the interplay of host cells and EV71. Furthermore, we found that a novel host factor, SLC35B2, played a dual role in regulating the overall sulfation comprising heparan sulfate sulfation and protein tyrosine sulfation, which are critical for EV71 entry. IMPORTANCE As the most important nonpolio neurotropic enterovirus lacking specific treatments, EV71 can transmit to the central nervous system, leading to severe and fatal neurological complications in infants and young children. The identification of new factors that facilitate or inhibit EV71 replication is crucial to uncover the mechanisms of viral infection and pathogenesis. To date, only a few host factors involved in EV71 infection have been characterized. Herein, we conducted a genome-wide CRISPR-Cas9 functional knockout (GeCKO) screen for the first time to study EV71 in HeLa cells. The screening results are presented as a ranked list of candidates, including 518 hits in the positive selection that facilitate EV71 replication and 1,044 hits in the negative selection that may be essential for cell growth and survival or for suppressing EV71 infection. We subsequently concentrated on the top three hits in the positive selection: SCARB2, SLC35B2, and B3GAT3. The knockout of any of these three genes confers strong resistance against EV71 infection. We confirmed that EV71 infection is codependent on two receptors, heparan sulfate and SCARB2. We also identified a host entry factor, SLC35B2, indirectly facilitating EV71 infection through regulation of the host cell sulfation, and determined a novel posttranslational modification, protein tyrosine sulfation existing in SCARB2. This study revealed that EV71 infectivity exhibits a significant positive correlation with the level of cellular sulfation regulated by SLC35B2. Due to the sulfation pathway being required for many distinct viruses, including but not limited to EV71 and respiratory syncytial virus (RSV), which were tested in this study, SLC35B2 represents a target of broad-spectrum antiviral therapy.

Enterovirus-71 utilizes small extracellular vesicles to cross the blood-brain barrier for infecting the central nervous system via transcytosis.

BACKGROUND: Central nervous system (CNS) infections caused by Enterovirus 71 (EV71) pose a serious threat to children, causing severe neurogenic complications and even fatality in some patients. However, the pathogenesis of EV71 infections in the CNS remains unclear. METHODS: An in vitro blood-brain barrier (BBB) model was constructed by coculturing brain microvascular endothelial cells (BMECs) and astrocytes in transwell inserts for simulating CNS infections. EV71 virions and small extracellular vesicles (sEVs) derived from EV71-infected cells (EV71-sEVs) were isolated from the cell culture supernatant by density gradient centrifugation. The BBB model was separately infected with EV71 virions and EV71-sEVs. The mechanism of crossing the BBB was determined by inhibiting the different endocytic modes. A murine model of EV71 infection was constructed for confirming the results of in vitro experiments. RESULTS: The EV71-sEVs containing viral components were endocytosed by BMECs and released on the abluminal side of the BBB model, where they infected the astrocytes without disrupting the BBB in the early stages of infection. The integrity of the tight junctions (TJs) between BMECs was breached via downregulation of PI3K/Akt signaling in the late stages of infection. CONCLUSIONS: EV71 utilized the circulating sEVs for infecting the CNS by crossing the BBB.

Enterovirus 71 enters human brain microvascular endothelial cells through an ARF6-mediated endocytic pathway.

Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in hand-foot-and-mouth disease. However, the mechanism responsible for how EV71 breaks through the blood-brain barrier to infect brain cells has yet to be elucidated. By performing a high-throughput small interfering RNA (siRNA) screening and validation, we found that the infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by caveolin, clathrin, and macropinocytosis but dependent on ADP-ribosylation factor 6 (ARF6), a small guanosinetriphosphate (GTP)-binding protein of the Ras superfamily. The specific siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by NAV-2729, a specific inhibitor of ARF6, in a dose-dependent manner. The subcellular analysis demonstrated the co-localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71 viral protein. Furthermore, ARF1, another small GTP-binding protein, was also found to participate in ARF6-mediated EV71 endocytosis. Murine experiments demonstrated that NAV-2729 significantly alleviated mortality caused by EV71 infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for drug development.

miR-342-5p targets CTNNBIP1 to promote enterovirus 71 replication.

OBJECTIVE: The aim of this research was to explore the role of miR-342-5p in EV71 replication. METHODS: Peritoneal injection of EV71 (107 TCID50/mL) at 50, 100, and 150 µL was conducted to infect 12-day-old suckling mice (n = 10 per group), and clinical scores and survival rates were recorded during a 6-day trial duration and followed by transcriptome sequencing of collected spinal cord tissues. The differential miRNAs and target genes of the infected and uninfected EV71 mice were analyzed. The miR-342 and CTNNBIP1 binding sites were detected using a dual luciferase reporter assay. Cell viability was detected by CCK-8. RT-qPCR, Western blot, immunofluorescence, and immunohistochemistry assays were conducted to detect VP1 protein levels. RESULTS: Transcriptome sequencing analyses know that the Wnt pathway played a role in EV71 infection, and the CTNNBIP1 gene in this pathway was the target gene of miR-342-5p. Whether in HMC3 cells or in the spinal cord tissue from the suckling mice, high levels of miR-342-5p markedly promoted EV71 VP1 mRNA and protein expression, elevated TNF-α, IL-6, and IL-10 levels, and inhibited IFN-ß levels. In addition, highly expressed miR-342-5p destroyed neuronal structure in spinal cord tissues and reduced the number of glial cells. Highly expressed CTNNBIP1 blocked the promotion of miR-342-5p in EV71 replication, and inhibited TNF-α, IL-6, and IL-10 levels, whereas elevated IFN-ß levels. This mechanism is that miR-342-5p can target the CTNNBIP1 3' UTR region, inhibit its expression and reduce its binding to CTNNB1, thus enhancing the interaction between CTNNB1 and TCF4 and activating the Wnt pathway-mediated type I interferon response. CONCLUSION: In nerve cells and tissues, the overexpression of miR-342-5p promoted the replication of EV71 and attenuated the innate immune response to antiviral disease via Wnt/CTNNB1 signaling pathway.

A review on current diagnostic tools and potential optical absorption spectroscopy for HFMD detection.

Hand, Foot, and Mouth Disease (HFMD) is an outbreak infectious disease that can easily spread among children under the age of five. The most common causative agents of HFMD are enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), but infection caused by EV71 is more associated with fatalities due to severe neurological disorders. The present diagnosis methods rely on physical examinations by the doctors and further confirmation by laboratories detection methods such as viral culture and polymerase chain reaction. Clinical signs of HFMD infection and other childhood diseases such as chicken pox, and allergies are similar, yet the genetics and pathogenicity of the viruses are substantially different. Thus, there is an urgent need for an early screening of HFMD using an inexpensive and user-friendly device that can directly detect the causative agents of the disease. This paper reviews current HFMD diagnostic methods based on various target types, such as nucleic acid, protein, and whole virus. This was followed by a thorough discussion on the emerging sensing technologies for HFMD detection, including surface plasmon resonance, electrochemical sensor, and surface enhanced Raman spectroscopy. Lastly, optical absorption spectroscopic method was critically discussed and proposed as a promising technology for HFMD screening and detection.

Molecular epidemiology and phylodynamic analysis of enterovirus 71 in Beijing, China, 2009-2019.

BACKGROUND: Enterovirus 71(EV71)-associated hand, foot and mouth disease (HFMD) decreased dramatically in Beijing from 2009 to 2019. This study was to investigate the epidemiological characteristics, evolutionary dynamics, geographic diffusion pathway, and other features of EV71 in Beijing, China. METHODS: We conducted a retrospective study of EV71-associated HFMD and its causative agent in Beijing, China, from 2009 to 2019. Phylogenetic and phylogeographic methods based on the EV71 genome were used to determine the evolution features, origin, and spatiotemporal dynamics. Positive selection sites in the VP1 gene were identified and exhibited in the tertiary structure. Bayesian birth-death skyline model was used to estimate the effective reproductive number (Re). RESULTS: EV71-associated HFMD decreased greatly in Beijing. From 2009 to 2019, EV71 strains prevalent in Beijing shared high homology in each gene segment and evolved with a rate of 4.99*10- 3 substitutions per site per year. The genetic diversity of EV71 first increased and peaked in 2012 and then decreased with fluctuations. The time to the most recent common ancestor (TMRCA) of EV71 in Beijing was estimated around 2003 when the EV71 strains were transmitted to Beijing from east China. Beijing played a crucial role in seeding EV71 to central China as well. Two residues (E145Q/G, A293S) under positive selection were detected from both the VP1 dataset and the P1 dataset. They were embedded within the loop of the VP1 capsid and were exposed externally. Mean Re estimate of EV71 in Beijing was about 1.007. CONCLUSION: In recent years, EV71 was not the primary causative agent of HFMD in Beijing. The low Re estimate of EV71 in Beijing implied that strategies for preventing and controlling HFMD were performed effectively. Beijing and east China played a crucial role in disseminating EV71 to other regions in China.

Does enterovirus 71 urge for effective vaccine control strategies? Challenges and current opinion.

Enterovirus 71 (EV71) is an infectious virus affecting all age groups of people around the world. It is one of the major aetiologic agents for HFMD (hand, foot and mouth disease) identified globally. It has led to many outbreaks and epidemics in Asian countries. Infection caused by this virus that can lead to serious psychological problems, heart diseases and respiratory issues in children younger than 10 years of age. Many studies are being carried out on the pathogenesis of the virus, but little is known. The host immune response and other molecular responses against the virus are also not clearly determined. This review deals with the interaction between the host and the EV71 virus. We discuss how the virus makes use of its proteins to affect the host's immunity and how the viral proteins help their replication. Additionally, we describe other useful resources that enable the virus to evade the host's immune responses. The knowledge of the viral structure and its interactions with host cells has led to the discovery of various drug targets for the treatment of the virus. Additionally, this review focusses on the antiviral drugs and vaccines developed by targeting various viral surface molecules during their infectious period. Furthermore, it is asserted that the improvement of prevailing vaccines will be the simplest method to manage EV71 infection swiftly. Therefore, we summarise numerous vaccines candidate for the EV71, such as the use of an inactivated complete virus, recombinant VP1 protein, artificial peptides, VLPs (viral-like particles) and live attenuated vaccines for combating the viral outbreaks promptly.

The Association of Helicobacter pylori Biofilm with Enterovirus 71 Prolongs Viral Viability and Survival.

The transition time during which a virus leaves its host and infects the next susceptible host is critical for virus survival. Enterovirus 71 (EV71) is stable in aqueous environments, but its molecular interactions with bacteria and their biofilms are not well-established. Helicobacter pylori is a highly successful gut bacterial pathogen, with its capacity to form biofilms being linked to its transmission. Given that both are gut-associated microbes, we hypothesized that biofilms formed by H. pylori may play a significant role in the survival of EV71 in the external environment. In this study, we examine the interactions of EV71 with the preformed biofilm of H. pylori to mimic its natural state in the environment. Immunofluorescence confocal microscopy and scanning electron microscopy revealed that EV71 particles persisted for up to 10 days when incubated with the H. pylori biofilm. Furthermore, the presence of the H. pylori biofilm significantly augmented viral viability, as verified through virus plaque assays. Interestingly, the viability of EV71 was dependent on the quantity of H. pylori biofilm formation. Thus, two H. pylori strains able to generate large amounts of biofilm could facilitate EV71 viability for up to 17 days, whereas two other H. pylori strains that produced moderate or low quantities of biofilm could not prolong virus viability. It is interesting that biofilm contains N-acetyl-glucosamine and glycosaminoglycan, and that EV71 has binding affinity to cell-surface heparan sulfate glycosaminoglycan, which acts as an EV71 attachment receptor. The synergistic ability of H. pylori biofilm to promote EV71 viability for extended periods implies that H. pylori biofilm may serve as an additional pathway of EV71 transmission.

Anti-α-glucosidase, Anti-proliferative and Anti-enterovirus 71 Activity of Secondary Metabolites Identified from Grifola Frondosa.

Grifola frondosa, an edible and medicinal resource, is widely used as functional foods worldwide. To explore bioactive compounds against α-glucosidase, human tumor cells and enterovirus 71 (EV71), eight compounds were isolated from G. frondosa by chromatographic column. Among the isolated compounds, heptadecanoic acid, uridine and adenosine exhibited potent inhibition activity against α-glucosidase, ergosterols and ergosterol-5,8-peroxide showed anti-proliferative activity on tumor cells, while ergosterol and methyl linoleate displayed inhibition against the replication of EV71. Also, to our knowledge, this is the first study to report that fatty acids isolated from G. frondosa show potent inhibition against α-glucosidase and EV71. Further molecular docking results revealed that the active compounds in G. frondosa form hydrogen bonding, hydrophobic interactive and π-stacking with the active sites on the surface of α-glucosidase, CASP3 and VP1 proteins, thus promoting the active compounds to combine with the target protein to form a stable complex, thus playing an antagonistic role. Our results could provide a new active compound and mode of action for G. frondosa to treat diabetes, cancer and EV71-infected patients.

Life-Threatening Enterovirus 71 Encephalitis in Unrelated Children with Autosomal Dominant TLR3 Deficiency.

PURPOSE: Enterovirus A71 (EV71) causes a broad spectrum of childhood diseases, ranging from asymptomatic infection or self-limited hand-foot-and-mouth disease (HFMD) to life-threatening encephalitis. The molecular mechanisms underlying these different clinical presentations remain unknown. We hypothesized that EV71 encephalitis in children might reflect an intrinsic host single-gene defect of antiviral immunity. We searched for mutations in the toll-like receptor 3 (TLR3) gene. Such mutations have already been identified in children with herpes simplex virus encephalitis (HSE). METHODS: We sequenced TLR3 and assessed the impact of the mutations identified. We tested dermal fibroblasts from a patient with EV71 encephalitis and a TLR3 mutation and other patients with known genetic defects of TLR3 or related genes, assessing the response of these cells to TLR3 agonist poly(I:C) stimulation and EV71 infection. RESULTS: Three children with EV71 encephalitis were heterozygous for rare mutations-TLR3 W769X, E211K, and R867Q-all of which were shown to affect TLR3 function. Furthermore, fibroblasts from the patient heterozygous for the W769X mutation displayed an impaired, but not abolished, response to poly(I:C). We found that TLR3-deficient and TLR3-heterozygous W769X fibroblasts were highly susceptible to EV71 infection. CONCLUSIONS: Autosomal dominant TLR3 deficiency may underlie severe EV71 infection with encephalitis. Human TLR3 immunity is essential to protect the central nervous system against HSV-1 and EV71. Children with severe EV71 infections, such as encephalitis in particular, should be tested for inborn errors of TLR3 immunity.

Distinct expression and clinical value of aquaporin 4 in children with hand, foot and mouth disease caused by enterovirus 71.

Brain dysfunction is a prerequisite for critical complications in children with hand, foot, and mouth disease (HFMD). Aquaporin 4 (AQP-4) may be involved in the pathological process of cerebral oedema and injury in children with severe and critical HFMD. This study aimed to assess the association of AQP-4 with the severity of enterovirus 71 (EV71)-associated HFMD. Children with EV71-infected HFMD were divided into a common group (clinical stage 1), a severe group (clinical stage 2), and a critical group (clinical stage 3) according to Chinese guidelines. The levels of AQP-4, interleukin-6 (IL-6), norepinephrine (NE), and neuron-specific enolase (NSE) before and after treatment were tested. Serum AQP-4, IL-6, NE, and NSE levels showed significant differences among the critical, severe, and common groups before and after treatment (P < 0.01). No significant differences in AQP-4 levels in cerebrospinal fluid (CSF) were observed between the critical and severe groups before and after treatment, but the CSF AQP-4 levels in these two groups were higher than those in the common group before treatment (P < 0.01). Serum AQP-4 levels, but not CSF AQP-4 levels, closely correlated with serum IL-6, NE, and NSE levels. These results suggest that the level of AQP-4 in serum, but not in CSF, is a candidate biomarker for evaluating the severity and prognosis of EV71-associated HFMD.

Enterovirus 71-induced autophagosome fusion with multivesicular bodies facilitates viral RNA packaging into exosomes.

Exosomes have been shown to release from cells infected by viruses and deliver viral particles, genomes, and other viral genetic elements to neighboring cells resulting in modulating host immune response. Our previous study demonstrated that exosomes released from Enterovirus 71 (EV71)-infected cells contained replication-competent EV71 RNA in complex with miR-146a, Ago2, and GW182, which can be successfully transferred to recipient/target cells to establish productive infection. However, the molecular mechanisms that control viral genome package into exosomes are still unclear. In this study, we showed that the EV71-induced autophagy response contributed to viral genome package into exosomes rather than process of exosomes biogenesis. Further study showed that the autophagosomes accumulation facilitated their fusion with MVBs, which resulted in EV71 RNA package into exosome vesicles. Moreover, prevention of autophagosomes-MVBs fusion could abolish this sorting of viral RNA into exosomes. Knockdown of GW182 or Ago2 could weaken the replication ability of exosomal EV71 RNA in recipient cells through decreasing the amount of miR-146a in exosomes, but did not affect the package of viral RNA into exosomes. Our findings strongly suggested that the accumulation of autophagosomes that were induced by EV71 infection play a key role on viral spreading through exosome vesicles.

Identification of the first C1 subgenotype of enterovirus 71 in the Chinese mainland in a retrospective study.

The C4 sub-genotype of Enterovirus 71 (EV71) has been identified as the most dominant sub-genotype circulating in the Chinese mainland since 1998. The circulation situation of EV71 before 1998 is not well established due to insufficient experimental data. The C1 subgenotype of EV71 has not yet been reported in the Chinese mainland by now. Based on the AFP surveillance system of the mainland of China, this study conducted a retrospective study of AFP cases for 1985-1999: a strain of EV-A71 C1 subgenotype was found. To our knowledge, this strain (SD92-41) is the first C1 sub-genotype reported in the Chinese mainland. This study demonstrates that the C1 gene subtype also appeared in the Chinese mainland, but it is unknown whether it is an imported or a local epidemic strain. With sufficient information known from retrospective studies, the source of the SD92-41 strain will be identified and the prevalence of EV-A71 in the Chinese mainland before 1998 will be clearer.

Prototypes virus of hand, foot and mouth disease infections and severe cases in Gansu, China: a spatial and temporal analysis.

BACKGROUND: Little research has been conducted on the spatio-temporal relationship between the severe cases and the enteroviruses infections of hand, foot and mouth disease (HFMD). This study aimed to investigate epidemic features and spatial clusters of HFMD incidence rates and assess the relationship between Enterovirus 71 (EV71) and Coxsackievirus A16 (CoxA16) and severe cases of HMFD in Gansu province, China. METHODS: Weekly county-specific data on HFMD between 1st January and 31st December 2018 were collected from the China Infectious Disease Information System (CIDIS), including enterovirus type (EV71 and CoxA16), severe and non-severe cases in Gansu province, China. Temporal risk [frequency index (α), duration index (ß) and intensity index (γ)] and spatial cluster analysis were used to assess epidemic features and identify high-risk areas for HFMD. Time-series cross-correlation function and regression model were used to explore the relationship between the ratios of two types of viruses (i.e. EV71/Cox16) (EC) and severe cases index (i.e. severe cases/non-severe cases) (SI) of HFMD. RESULTS: Some counties in Dingxi City, Gansu were identified as a hot spot for the temporal risk indices. Time-series cross-correlation analysis showed that SI was significantly associated with EC (r = 0.417, P < 0.05) over a 4-week time lag. The regression analysis showed that SI was positively associated with EC (ß = 0.04, 95% confidence interval (CI) 0.02-0.06). CONCLUSION: The spatial patterns of HFMD incidence were associated with enteroviruses in Gansu. The research suggested that the EC could be considered a potential early warning sign for predicting severe cases of HFMD in Gansu province.

Anemoside B4 inhibits enterovirus 71 propagation in mice through upregulating 14-3-3 expression and type I interferon responses.

Enterovirus 71 (EV71) is the major pathogens of human hand, foot, and mouth disease (HFMD). EV71 efficiently escapes innate immunity responses of the host to cause infection. At present, no effective antiviral drugs for EV71 are available. Anemoside B4 (B4) is a natural saponin isolated from the roots of Pulsatilla chinensis (Bunge) Regel. P. chinensis extracts that shows a wide variety of biological activities. In this study, we investigated the antiviral activities of B4 against EV71 both in cell culture and in suckling mice. We showed that B4 (12.5-200 µM) dose dependently increased the viability of EV71-infected RD cells with an IC50 value of 24.95 ± 0.05 µM against EV71. The antiviral activity of B4 was associated with enhanced interferon (IFN)-ß response, since knockdown of IFN-ß abolished its antiviral activity. We also confirmed that the enhanced IFN response was mediated via activation of retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) pathway, and it was executed by upregulation of 14-3-3 protein, which disrupted the interaction between yes-associated protein (YAP) and interferon regulatory factor 3 (IRF3). By using amino acids in cell culture (SILAC)-based proteomics profiling, we identified the Hippo pathway as the top-ranking functional cluster in B4-treated EV71-infected cells. In vivo experiments were conducted in suckling mice (2-day-old) infected with EV71 and subsequently B4 (200 mg · kg-1 · d-1, i.p.) was administered for 16 days. We showed that B4 administration effectively suppressed EV71 replication and improved muscle inflammation and limb activity. Meanwhile, B4 administration regulated the expressions of HFMD biomarkers IL-10 and IFN-γ, attenuating complications of EV71 infection. Collectively, our results suggest that B4 could enhance the antiviral effect of IFN-ß by orchestrating Hippo and RLRs pathway, and B4 would be a potential lead compound for developing an anti-EV71 drug.

Spatial-temporal-demographic and virological changes of hand, foot and mouth disease incidence after vaccination in a vulnerable region of China.

BACKGROUND: The enterovirus 71 (EV-A71) vaccine has been used in Hefei for several years, and the epidemiological significance of vaccination in this area is unclear. We aims to explore the spatial-temporal-demographic and virological changes of hand, foot and mouth disease (HFMD) after vaccination in China. METHODS: The data for HFMD from 2012 to 2020 were downloaded with the help of HFMD reporting system of Hefei Center for Disease Control and Prevention and combined with the EV-A71 vaccination status in Hefei. The study defined the period between 2012 to 2016 as the pre-vaccination period and explored the effect of vaccination on the incidence of HFMD by comparing the changes of HFMD before and after vaccination in terms of spatial, temporal, demographic and virological aspects. RESULTS: During the study period, a higher incidence occurred in urban area and the random distribution changed to a slight cluster after vaccination. HFMD incidence had inconsistent seasonality over years, with one or two incidence peaks in varying years. The morbidity decreased from 215.22/105 in 2012-2016 to 179.81/105 in 2017-2020 (p < 0.001). Boys, 0-4 years old children and Scattered children were more susceptible to HFMD compared with the others, the proportions decreased after vaccination except in Scattered children. The main pathogenic enterovirus gradually changed from EV-A71 to Other Enteroviruses, especially coxsackieviruses A6 (CV-A6) after the implementation of EV-A71 vaccination. CONCLUSIONS: The EV-A71 vaccine was effective in reducing the incidence of HFMD and changing the spatial, temporal, demographic, and virological characteristic. These changes should be considered during the vaccination implementation to further reduce the disease burden of HFMD.

The Bottlenecks of Preparing Virus Particles by Size Exclusion for Antibody Generation.

Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 106 and 1.3 × 107 transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.