Human Hand, Foot, And Mouth Disease Research Articles

Highly sensitive and rapid identification of coxsackievirus A16 based on reverse transcription multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor assay.

One of the main pathogens responsible for human hand, foot, and mouth disease (HFMD), coxsackievirus A16, has put young children's health at danger, especially in countries in the Asia-Pacific region. Early quick identification is essential for the avoidance and control of the disorder since there are no vaccinations or antiviral medications available to prevent and manage CVA16 infection. Here, we describe the creation of an easy, speedy, and accurate CVA16 infection detection approach using lateral flow biosensors (LFB) and reverse transcriptionmultiple cross displacement amplification (RT-MCDA). A group of 10 primers was developed for the RT-MCDA system in order to amplify the genes in an isothermal amplification device while targeting the highly conserved region of the CVA16 VP1 gene. Then, without requiring any extra tools, RT-MCDA amplification reaction products might well be detected by visual detection reagent (VDR) and LFB. The outcomes showed that 64°C within 40 min was the ideal reaction setting for the CVA16-MCDA test. Target sequences with <40 copies might be found using the CVA16-MCDA. There was no cross-reaction among CVA16 strains and other strains. The findings demonstrated that the CVA16-MCDA test could promptly and successfully identify all of the CVA16-positive (46/220) samples identified by the traditional real-time quantitative polymerase chain reaction (qRT-PCR) assays for 220 clinical anal swab samples. The whole process, such as the processing of the sample (15 min), the MCDA reaction (40 min), and the documenting of the results (2 min), could be finished in 1 h. The CVA16-MCDA-LFB assay, which targeted the VP1 gene, was an efficient, simple, and highly specific examination that might be used extensively in rural regions' basic healthcare institutions and point-of-care settings.

Development and evaluation of an inactivated coxsackievirus A16 vaccine in gerbils

ABSTRACT Coxsackievirus A16 (CVA16) is one of the major pathogens responsible for human hand, foot, and mouth disease (HFMD), which has threatened the health of young children, particularly in Asia-Pacific nations. Vaccination is an effective strategy for protecting children from CVA16 infection. However, there is currently no licensed CVA16 vaccine for use in humans. In this study, we isolated a high-growth CVA16 virus strain in MRC-5 cells and developed an MRC-5-adapted vaccine candidate strain termed CVA16-393 via two rounds of plaque purification. The CVA16-393 strain was grouped into the B1b subgenotype and grew to a titre of over 107 TCID50/ml in MRC-5 cells. The VP1 gene region of this strain, which contains the major neutralizing epitopes, displayed high stability during serial passages. The inactivated whole-virus vaccine produced by the CVA16-393 strain induced an effective neutralizing antibody response in Meriones unguiculatus (gerbils) after two doses of intraperitoneal inoculation. One week after the booster immunization, the geometric mean titres of the neutralizing antibodies for the 10246, 40812TXT, 11203SD, TJ-224 and CA16-194 strains from different regions of China were 137.8, 97.8, 113.4, 64.1 and 122.3, respectively. A CVA16 vaccine dose above 25 U was also able to provide 100% cross-protection against lethal challenges with these five clinical strains in gerbils. Immunization at a one-week interval could maintain a high level of neutralizing antibody titres for at least 8 weeks. Thus, the vaccine produced by this CVA16-393 strain might be promising.

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)-β response, since knockdown of IFN-β 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 administeredfor 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-β by orchestrating Hippo and RLRs pathway, and B4 would be a potential lead compound for developing an anti-EV71 drug.

A hSCARB2-transgenic mouse model for Coxsackievirus A16 pathogenesis

BackgroundCoxsackievirus A16 (CA16) is one of the neurotropic pathogen that has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD), but its pathogenesis is not yet clear. The limited host range of CA16 make the establishment of a suitable animal model that can recapitulate the neurological pathology observed in human HFMD more difficult. Because the human scavenger receptor class B, member 2 (hSCARB2) is a cellular receptor for CA16, we used transgenic mice bearing human SCARB2 and nasally infected them with CA16 to study the pathogenicity of the virus.MethodsCoxsackievirus A16 was administered by intranasal instillation to groups of hSCARB2 transgenic mice and clinical signs were observed. Sampled at different time-points to document and characterize the mode of viral dissemination, pathological change and immune response of CA16 infection.ResultsWeight loss and virus replication in lung and brain were observed in hSCARB2 mice infected with CA16, indicating that these animals could model the neural infection process. Viral antigens were observed in the alveolar epithelia and brainstem cells. The typical histopathology was interstitial pneumonia with infiltration of significant lymphocytes into the alveolar interstitial in lung and diffuse punctate hemorrhages in the capillaries of the brainstem. In addition, we detected the expression levels of inflammatory cytokines and detected high levels of interleukin IL-1β, IL-6, IL-18, and IFN-γ in nasal mucosa, lungs and brain tissues.ConclusionsThe hSCARB2-transgenic mice can be productively infected with CA16 via respiratory route and exhibited a clear tropism to lung and brain tissues, which can serve as a model to investigate the pathogenesis of CA16 associated respiratory and neurological disease.

Efficacy of an inactivated bivalent vaccine for enterovirus 71 and coxsackievirus A16 in mice immunized intradermally

Human hand, foot, and mouth disease (HFMD), an important infectious disease in children, is caused mainly by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In this study, a bivalent inactivated EV71/CA16 vaccine is developed and evaluated in immunized BALB/c mice injected through the intradermal route. Q-RT-PCR detection of the mRNA of immune signal molecules in local epithelial tissues inoculated with the vaccine indicates activation of innate immunity, which includes upregulation of immune-related chemokines, interferons and CD molecules. Further, the finding that neutralizing antibodies and specific T cellular responses were elicited in adult mice after two immunizations with the vaccine at a 28-day interval, which endowed offspring mice to defend a viral challenge, suggests the successful induction of specific protective antiviral immunity. All these data suggest that immunization with this bivalent EV71/CA16 vaccine via the intradermal route elicits effective immunity against EV71 and CA16 infection.

Design, Synthesis, and Evaluation of Novel Enterovirus 71 Inhibitors as Therapeutic Drug Leads for the Treatment of Human Hand, Foot, and Mouth Disease.

Human hand, foot, and mouth disease (HFMD) is a serious public health threat with high infection rates in children and infants who reside in Asia and the Pacific regions, and no effective drugs are currently available. Enterovirus 71 (EV71) and coxsackievirus A16 are the major etiological pathogens. Based on an essential hydrophobic pocket on the viral capsid protein VP1, we designed and synthesized a series of small molecular weight compounds as inhibitors of EV71. A potential drug candidate named NLD-22 exhibited excellent antiviral activity (with an EC50 of 5.056 nM and a 100% protection rate for mice at a dose of 20 mg/kg) and low toxicity. NLD-22 had a favorable pharmacokinetic profile. High-resolution cryo-electron microscopy structural analysis confirmed NLD-22 bound to the hydrophobic pocket in VP1 to block viral infection. In general, NLD-22 was indicated to be a promising potential drug candidate for the treatment of HFMD.

Role of innate lymphoid cells and dendritic cells in intradermal immunization of the enterovirus antigen

Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are the major pathogens of human hand, foot, and mouth disease (HFMD). In our previous study, intramuscular immunization with the inactivated EV71 vaccine elicited effective immunity, while immunization with the inactivated CA16 vaccine did not. In this report, we focused on innate immune responses elicited by inactivated EV71 and CA16 antigens administered intradermally or intramuscularly. The distributions of the EV71 and CA16 antigens administered intradermally or intramuscularly were not obviously different, but the antigens were detected for a shorter period of time when administered intradermally. The expression levels of NF-κB pathway signaling molecules, which were identified as being capable of activating DCs, ILCs, and T cells, were higher in the intradermal group than in the intramuscular group. Antibodies for the EV71 and CA16 antigens colocalized with ILCs and DCs in skin and muscle tissues under fluorescence microscopy. Interestingly, ILC colocalization decreased over time, while DC colocalization increased over time. ELISpot analysis showed that coordination between DCs and ILCs contributed to successful adaptive immunity against vaccine antigens in the skin. EV71 and/or CA16 antigen immunization via the intradermal route was more capable of significantly increasing neutralizing antibody titers and activating specific T cell responses than immunization via the intramuscular route. Furthermore, neonatal mice born to mothers immunized with the EV71 and CA16 antigens were 100% protected against wild-type EV71 or CA16 viral challenge. Together, our results provide new insights into the development of vaccines for HFMD.

The immunogenicity and protection effect of an inactivated coxsackievirus A6, A10, and A16 vaccine against hand, foot, and mouth disease

Coxsackievirus belongs to the Enterovirus genus of the Picornaviridae family and is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD). Historically, outbreaks of HFMD have mainly been caused by enterovirus 71 and coxsackievirus A16. Recently, coxsackieviruses A6 and A10 have been associated with increased occurrences of sporadic HFMD cases and outbreak events globally. In this study, the immunogenicity of coxsackieviruses A6, A10, and A16 (CA6, CA10, and CA16), which were inactivated by formalin or β-propiolactone (BPL) under different conditions, was evaluated as multivalent vaccine candidates. CA6 induced similar immune responses with both inactivation methods, and the immune efficacy of CA10 and CA16 was better following inactivation with BPL than with formalin. There was no sufficient cross-reactivity or cross-protectivity against heterologous strains in groups vaccinated with the BPL-inactivated (BI) monovalent vaccine. Sufficient neutralizing antibody and cell-mediated immune responses were induced in the BI-trivalent vaccinated group. These findings suggest that BI-CA6, CA10, and CA16 are potential multivalent vaccine candidates and that a multivalent vaccine is needed to control HFMD. The coxsackievirus multivalent vaccine could be useful for the development of effective HFMD vaccines.

Hsc70 regulates the IRES activity and serves as an antiviral target of enterovirus A71 infection

Enterovirus A71 (EV-A71) is a small positive-stranded RNA virus that causes human hand, foot and mouth disease (HFMD) and fatal neurological disorders in some cases without effective treatment. Here we show that heat shock cognate protein 70 (Hsc70), a molecular chaperone, displays pivotal role in viral infections. Knockdown of Hsc70 significantly suppresses viral replication evidenced by reducing not only the level of both viral replication intermediates (negative stranded RNA) and viral genomic RNA (positive stranded RNA), but also the level of viral protein expression; whereas ectopic expression of Hsc70 markedly promotes viral replication. Interestingly, depletion of Hsc70 decreases the IRES activity of EV-A71, and the ectopic expression of Hsc70 enhances the IRES activity accordingly. Further study shows that Hsc70 binds viral genomic RNA but does not directly interact with IRES. Moreover, we reveal that Hsc70 interacts with 2A protease and promotes eIF4G cleavage. More importantly, Hsc70 inhibitor Ver-155008 significantly protects cytopathic effects from EV-A71 infection and inhibits both IRES activity and viral reproduction in a dose-dependent manner. The cell viability assay shows that the IC50 and CC50 are 2.01 μM and 47.67 μM, respectively. These results demonstrate not only an important mechanism of Hsc70 in facilitating EV-A71 replication, but also a target for antiviral drug development.

Inhibition of enterovirus 71 replication by an α-hydroxy-nitrile derivative NK-1.9k

Enterovirus 71 (EV71) is one of the major etiological agents of human hand-foot-and-mouth disease (HFMD) worldwide. EV71 infection in young children and people with immunodeficiency causes severe symptoms with a high fatality rates. However, there is still no approved drugs to treat such infections. Based on our previous report of a peptide-aldehyde anti-EV71 protease, we present here a highly specific α-hydroxy-nitrile derivative NK-1.9k, which inhibited the proliferation of multiple EV71 strains and coxsackievirus A16 (CVA16) in various cells with EC50 of 37.0 nM with low cytotoxicity (CC50 > 200 μM). The hydroxy-nitrile covalent warhead conferred NK-1.9k high potency and selectivity to interact with the cysteine residue of the active site of the viral protease. We also documented the resistance to NK-1.9k with a N69S mutation in EV71 3Cpro. The combination of NK-1.9k and EV71 polymerase or entry inhibitors produced strong synergistic antiviral effects. Collectively, our findings suggest our compounds can potentially be developed as drugs for the treatment of HFMD.

Coxsackievirus A16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation.

Coxsackievirus A16 (CA16) is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD) in the Asia-pacific region. Although CA16 infections are generally mild, severe neurological manifestations or even death has been reported. Studies on CA16 pathogenesis and vaccine development are severely hampered because the small animal models that are currently available show major limitations. In this study, gerbils (Meriones unguiculatus) were investigated for their suitability as an animal model to study CA16 pathogenesis and vaccine development. Our results showed that gerbils up to the age of 21 days were fully susceptible to CA16 and all died within five days post-infection. CA16 showed a tropism towards the skeletal muscle, spinal cord and brainstem of gerbils, and severe lesions, including necrosis, were observed. In addition, an inactivated CA16 whole-virus vaccine administrated to gerbils was able to provide full protection to the gerbils against lethal doses of CA16 strains. These results demonstrate that gerbils are a suitable animal model to study CA16 infection and vaccine development.

A Conserved Inhibitory Mechanism of a Lycorine Derivative against Enterovirus and Hepatitis C Virus.

Enterovirus 71 (EV71) (Picornaviridae family) and hepatitis C virus (HCV) (Flaviviridae family) are the causative agents of human hand, foot, and mouth disease (HFMD) and hepatitis C, resulting in a severe pandemic involving millions of infections in the Asia-Pacific region and worldwide. The great impact of EV71 and HCV on public health highlights the need to further our understanding of the biology of these two viruses and develop effective therapeutic antivirals. Here, we evaluated a total of 32 lycorine derivatives and demonstrated that 1-acetyllycorine suppressed the proliferation of multiple strains of EV71 in various cells. The results of the drug resistance analysis revealed that 1-acetyllycorine targeted a phenylalanine (F76) in EV71 2A protease (2A(pro)) to stabilize the conformation of a unique zinc finger. Most interestingly, the zinc binding site in EV71 2A(pro) is the exclusive homolog of HCV NS3 among all viruses. Further analysis revealed that 1-acetyllycorine also inhibits HCV with high efficacy, and the mutation on R118 in HCV NS3, which corresponds to F76 in EV71 2A(pro), confers the resistance of HCV to 1-acetyllycorine. These results revealed a conserved mechanism of 1-acetyllycorine against EV71 and HCV through targeting viral proteases. We also documented the significant synergistic anti-EV71 and anti-HCV effects of 1-acetyllycorine with reported inhibitors, supporting potential combination therapy for the treatment of EV71 and HCV infections.

Comparative Study of the Cytokine/Chemokine Response in Children with Differing Disease Severity in Enterovirus 71-Induced Hand, Foot, and Mouth Disease

BackgroundEnterovirus 71 (EV71) infection can lead to a rapidly progressing, life-threatening, and severe neurological disease in young children, including the development of human hand, foot, and mouth disease (HFMD). This study aims to further characterize the specific immunological features in EV71–mediated HFMD patients presenting with differing degrees of disease severity.MethodologyComprehensive cytokine and chemokine expression were broadly evaluated by cytokine antibody array in EV71–infected patients hospitalized for HFMD compared to Coxsackievirus A16-infected patients and age-matched healthy controls. More detailed analysis using Luminex-based cytokine bead array was performed in EV71–infected patients stratified into diverse clinic outcomes. Additionally, immune cell frequencies in peripheral blood and EV71–specific antibodies in plasma were also examined.Principal FindingsExpression of several cytokines and chemokines were significantly increased in plasma from EV71–infected patients compared to healthy controls, which further indicated that: (1) GM-CSF, MIP-1β, IL-2, IL-33, and IL-23 secretion was elevated in patients who rapidly developed disease and presented with uncomplicated neurological damage; (2) G-CSF and MCP-1 were distinguishably secreted in EV71 infected very severe patients presenting with acute respiratory failure; (3) IP-10, MCP-1, IL-6, IL-8, and G-CSF levels were much higher in cerebrospinal fluid than in plasma from patients with neurological damage; (4) FACS analysis revealed that the frequency of CD19+HLADR+ mature B cells dynamically changed over time during the course of hospitalization and was accompanied by dramatically increased EV71–specific antibodies. Our data provide a panoramic view of specific immune mediator and cellular immune responses of HFMD and may provide useful immunological profiles for monitoring the progress of EV71–induced fatal neurological symptoms with acute respiratory failure.

A Neonatal Mouse Model of Coxsackievirus A16 for Vaccine Evaluation

To evaluate vaccine efficacy in protecting against coxsackievirus A16 (CA16), which causes human hand, foot, and mouth disease (HFMD), we established the first neonatal mouse model. In this article, we report data concerning CA16-induced pathological changes, and we demonstrate that anti-CA16 antibody can protect mice against lethal challenge and that the neonatal mouse model could be used to evaluate vaccine efficacy. To establish a mouse model, a BJCA08/CA16 strain (at 260 50% lethal doses [LD(50)]) was isolated from a patient and used to intracerebrally (i.c.) inoculate neonatal mice. The infection resulted in wasting, hind-limb paralysis, and even death. Pathological examination and immunohistochemistry (IHC) staining indicated that BJCA08 had a strong tropism to muscle and caused severe necrosis in skeletal and cardiac muscles. We then found that BJCA08 pretreated with goat anti-G10/CA16 serum could significantly lose its lethal effect in neonatal mice. When the anti-G10 serum was intraperitoneally (i.p.) injected into the neonatal mice and, within 1 h, the same mice were intracerebrally inoculated with BJCA08, there was significant passive immunization protection. In a separate experiment, female mice were immunized with formaldehyde-inactivated G10/CA16 and BJCA08/CA16 and then allowed to mate 1 h after the first immunization. We found that there was significant protection against BJCA08 for neonatal mice born to the immunized dams. These data demonstrated that anti-CA16 antibody may block virus invasion and protect mice against lethal challenge, and that the neonatal mouse model was a viable tool for evaluating vaccine efficacy.