Enterovirus virus-like-particle and inactivated poliovirus vaccines do not elicit substantive cross-reactive antibody responses.

Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations.

VP1 codon deoptimization and high-fidelity substitutions in 3D polymerase as potential vaccine strategies for eliciting immune responses against enterovirus A71.

Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-γ, IL-4, IL-6, and TNF-α) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection.IMPORTANCEEV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71.

Identification of four neutralizing antigenic sites on the enterovirus D68 capsid.

IMPORTANCE: Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 ßB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.

Rhinovirus-induced epithelial RIG-I inflammasome suppresses antiviral immunity and promotes inflammation in asthma and COVID-19.

Rhinoviruses and allergens, such as house dust mite are major agents responsible for asthma exacerbations. The influence of pre-existing airway inflammation on the infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. We analyse mechanisms of response to viral infection in experimental in vivo rhinovirus infection in healthy controls and patients with asthma, and in in vitro experiments with house dust mite, rhinovirus and SARS-CoV-2 in human primary airway epithelium. Here, we show that rhinovirus infection in patients with asthma leads to an excessive RIG-I inflammasome activation, which diminishes its accessibility for type I/III interferon responses, leading to their early functional impairment, delayed resolution, prolonged viral clearance and unresolved inflammation in vitro and in vivo. Pre-exposure to house dust mite augments this phenomenon by inflammasome priming and auxiliary inhibition of early type I/III interferon responses. Prior infection with rhinovirus followed by SARS-CoV-2 infection augments RIG-I inflammasome activation and epithelial inflammation. Timely inhibition of the epithelial RIG-I inflammasome may lead to more efficient viral clearance and lower the burden of rhinovirus and SARS-CoV-2 infections.

Construction of a Vero Cell Line Expressing Human ICAM1 for the Development of Rhinovirus Vaccines.

Human rhinoviruses (HRVs) are small non-enveloped RNA viruses that belong to the Enterovirus genus within the Picornaviridae family and are known for causing the common cold. Though symptoms are generally mild in healthy individuals, the economic burden associated with HRV infection is significant. A vaccine could prevent disease. The Vero-cell-based viral vaccine platform technology was considered for such vaccine development. Unfortunately, most HRV strains are unable to propagate on Vero cells due to a lack of the major receptor of HRV group A and B, intercellular adhesion molecule (ICAM1, also known as CD54). Therefore, stable human ICAM1 expressing Vero cell clones were generated by transfecting the ICAM1 gene in Vero cells and selecting clones that overexpressed ICAM1 on the cell surface. Cell banks were made and expression of ICAM1 was stable for at least 30 passages. The Vero_ICAM1 cells and parental Vero cells were infected with four HRV prototypes, B14, A16, B37 and A57. Replication of all four viruses was detected in Vero_ICAM1, but not in the parental Vero cells. Altogether, Vero cells expressing ICAM1 could efficiently propagate the tested HRV strains. Therefore, ICAM1-expressing cells could be a useful tool for the development and future production of polyvalent HRV vaccines or other viruses that use ICAM1 as a receptor.

Chemokine PF4 Inhibits EV71 and CA16 Infections at the Entry Stage.

Platelet factor 4 (PF4) or the CXC chemokine CXCL4 is the most abundant protein within the α-granules of platelets. Previous studies found that PF4 regulates infections of several viruses, including HIV-1, H1N1, hepatitis C virus (HCV), and dengue virus. Here, we show that PF4 is an inhibitor of enterovirus A71 (EV71) and coxsackievirus A16 (CA16) infections. The secreted form of PF4 from transfected cells or soluble purified PF4 from Escherichia coli, even lacking signal peptide affected secretion, obviously inhibited the propagation of EV71 and CA16. Mechanistically, we demonstrated that PF4 blocked the entry of the virus into the host cells by interactions with VP3 proteins of EV71/CA16 and the interaction with SCARB2 receptor-mediated EV71 and CA16 endocytosis. As expected, the incubation of anti-PF4 antibody with PF4 blocked PF4 inhibition on EV71 and CA16 infections further supported the above conclusion. Importantly, pretreatment of EV71 viruses with PF4 significantly protected the neonatal mice from EV71 lethal challenge and promoted the survival rate of infected mice. PF4 derived from natural platelets by EV71/CA16 activation also presented strong inhibition on EV71 and CA16. In summary, our study identified a new host factor against EV71 and CA16 infections, providing a novel strategy for EV71 and CA16 treatment. IMPORTANCE The virus's life cycle starts with binding to cell surface receptors, resulting in receptor-mediated endocytosis. Targeting the entry of the virus into target cells is an effective strategy to develop a novel drug. EV71 and CA16 are the major pathogens that cause hand, foot, and mouth disease (HFMD) outbreaks worldwide since 2008. However, the treatment of EV71 and CA16 infections is mainly symptomatic because there is no approved drug. Therefore, the underlying pathogenesis of EV71/CA16 and the interaction between host-EV71/CA16 need to be further investigated to develop an inhibitor. Here, we identified PF4 as a potent entry inhibitor of EV71 and CA16 via binding to VP3 proteins of EV71 and CA16 or binding to receptor SCARB2. In the EV71 infection model, PF4 protected mice from EV71 lethal challenge and promoted the survival rate of EV71-infected mice. Our study suggests that PF4 represents a potential candidate host factor for anti-EV71 and CA16 infections.

Neurotropic EV71 causes encephalitis by engaging intracellular TLR9 to elicit neurotoxic IL12-p40-iNOS signaling.

Brainstem encephalitis, a manifestation of severe enterovirus 71 (EV71) infection, is an acute excessive inflammatory response. The mechanisms underlying its development remain poorly understood. Usually neurotropic viruses trigger acute host immune response by engaging cell surface or intracellular receptors. Here, we show that EV71 engagement with intracellular receptor TLR9 elicits IL-12p40-iNOS signaling causing encephalitis in mice. We identified IL-12p40 to be the only prominent cytokine-induced at the early infection stage in the brainstem of mice subjected to a lethal dose of EV71. The upregulated IL-12p40 proteins were expressed in glial cells but not neuronal cells. To better understand the role of IL-12p40 in severe EV71 infection, we treated the EV71-infected mice with an antibody against IL-12p40 and found the mortality rate, brainstem inflammation, and gliosis to be markedly reduced, suggesting that the acute IL-12p40 response plays a critical role in the pathogenesis of brainstem encephalitis. Mechanistically, intracellular TLR9 was found essential to the activation of the IL-12p40 response. Blocking TLR9 signaling with CpG-ODN antagonist ameliorated IL-12p40 response, brainstem inflammation, and limb paralysis in mice with EV71-induced encephalitis. We further found the glial IL-12p40 response might damage neurons by inducing excess production of neurotoxic NO by iNOS. Overall, EV71 engagement with intracellular TLR9 was found to elicit a neurotoxic glial response via IL12p40-iNOS signaling contributing to the neurological manifestation of EV71 infection. This pathway could potentially be targeted for the treatment of brainstem encephalitis.

Global prevalence and case fatality rate of Enterovirus D68 infections, a systematic review and meta-analysis.

A substantial amount of epidemiological data has been reported on Enterovirus D68 (EV-D68) infections after the 2014 outbreak. Our goal was to map the case fatality rate (CFR) and prevalence of current and past EV-D68 infections. We conducted a systematic review (PROSPERO, CRD42021229255) with published articles on EV-68 infections in PubMed, Embase, Web of Science and Global Index Medicus up to January 2021. We determined prevalences using a model random effect. Of the 4,329 articles retrieved from the databases, 89 studies that met the inclusion criteria were from 39 different countries with apparently healthy individuals and patients with acute respiratory infections, acute flaccid myelitis and asthma-related diseases. The CFR estimate revealed occasional deaths (7/1353) related to EV-D68 infections in patients with severe acute respiratory infections. Analyses showed that the combined prevalence of current and past EV-D68 infections was 4% (95% CI = 3.1-5.0) and 66.3% (95% CI = 40.0-88.2), respectively. The highest prevalences were in hospital outbreaks, developed countries, children under 5, after 2014, and in patients with acute flaccid myelitis and asthma-related diseases. The present study shows sporadic deaths linked to severe respiratory EV-D68 infections. The study also highlights a low prevalence of current EV-D68 infections as opposed to the existence of EV-D68 antibodies in almost all participants of the included studies. These findings therefore highlight the need to implement and/or strengthen continuous surveillance of EV-D68 infections in hospitals and in the community for the anticipation of the response to future epidemics.

Protein Kinase B2 (PKB2/AKT2) Is Essential for Host Protection in CVB3-Induced Acute Viral Myocarditis.

Protein kinase B2 (AKT2) is involved in various cardiomyocyte signaling processes, including those important for survival and metabolism. Coxsackievirus B3 (CVB3) is one of the most common pathogens that cause myocarditis in humans. The role of AKT2 in CVB3 infection is not yet well understood. We used a cardiac-specific AKT2 knockout (KO) mouse to determine the role of AKT2 in CVB3-mediated myocarditis. CVB3 was injected intraperitoneally into wild-type (WT) and KO mice. The mice's survival rate was recorded: survival in KO mice was significantly decreased compared with WT mice (WT vs. KO: 73.3 vs. 27.1%). Myocardial damage and inflammation were significantly increased in the hearts of KO mice compared with those of WT mice. Moreover, from surface ECG, AKT2 KO mice showed a prolonged atria and ventricle conduction time (PR interval, WT vs. KO: 47.27 ± 1.17 vs. 64.79 ± 7.17 ms). AKT2 deletion induced severe myocarditis and cardiac dysfunction due to CVB3 infection. According to real-time PCR, the mRNA level of IL-1, IL-6, and TNF-α decreased significantly in KO mice compared with WT mice on Days 5 after infection. In addition, innate immune response antiviral effectors, Type I interferon (interferon-α and ß), and p62, were dramatically suppressed in the heart of KO mice. In particular, the adult cardiac myocytes isolated from the heart showed high induction of TLR4 protein in KO mice in comparison with WT. AKT2 deletion suppressed the activation of Type I interferon and p62 transcription in CVB3 infection. In cardiac myocytes, AKT2 is a key signaling molecule for the heart from damage through the activation of innate immunity during acute myocarditis.

Transcriptomic Profiling Reveals a Role for TREM-1 Activation in Enterovirus D68 Infection-Induced Proinflammatory Responses.

Increasing cases related to the pathogenicity of Enterovirus D68 (EV-D68) have made it a growing worldwide public health concern, especially due to increased severe respiratory illness and acute flaccid myelitis (AFM) in children. There are currently no vaccines or medicines to prevent or treat EV-D68 infections. Herein, we performed genome-wide transcriptional profiling of EV-D68-infected human rhabdomyosarcoma (RD) cells to investigate host-pathogen interplay. RNA sequencing and subsequent experiments revealed that EV-D68 infection induced a profound transcriptional dysregulation of host genes, causing significantly elevated inflammatory responses and altered antiviral immune responses. In particular, triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in highly activated TREM-1 signaling processes, acting as an important mediator in EV-D68 infection, and it is related to upregulation of interleukin 8 (IL-8), IL-6, IL-12p70, IL-1ß, and tumor necrosis factor alpha (TNF-α). Further results demonstrated that NF-κB p65 was essential for EV-D68-induced TREM-1 upregulation. Moreover, inhibition of the TREM1 signaling pathway by the specific inhibitor LP17 dampened activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade, suggesting that TREM-1 mainly transmits activation signals to phosphorylate p38 MAPK. Interestingly, treatment with LP17 to inhibit TREM-1 inhibited viral replication and infection. These findings imply the pathogenic mechanisms of EV-D68 and provide critical insight into therapeutic intervention in enterovirus diseases.

Inborn errors of TLR3- or MDA5-dependent type I IFN immunity in children with enterovirus rhombencephalitis.

Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-ß in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively.

Structural basis for neutralization of enterovirus.

Outbreaks of enteroviral infections are associated with morbidity and mortality in susceptible individuals worldwide. There are still no antiviral drugs or vaccines against most circulating enteroviruses. Antibody-mediated immunity is crucial for preventing and limiting enteroviral infections. In this review, we focus on enteroviruses that continue to cause endemics in recent years, such as rhinovirus, enterovirus A71, coxsackievirus, and echovirus, and introduce a structural understanding of the mechanisms of virus neutralization. The mechanisms by which virus-specific antibodies neutralize enteroviruses have been explored not only through study of viral structures, but also through understanding virus-antibody interactions at the amino acid level. Neutralizing epitopes are predominantly mapped on the canyon northern rim, canyon inner surface, canyon southern rim, and twofold and threefold plateaus of the capsid, where surface-exposed loops are located. This review also describes recent progress in deciphering the virus-receptor complex and structural rearrangements involved in the uncoating process, providing insight into plausible virus neutralization mechanisms.

Clinical and Immune Responses of Peripheral Chemical Sympathectomy in Enterovirus 71 Infection.

The activation of the sympathetic nervous system, release of norepinephrine (NE), and adrenergic receptor signaling participate in and regulate the complicated enterovirus 71 (EV71) brainstem encephalitis (BE). The neurotoxin 6-hydroxydopamine (6-OHDA) selectively ablates sympathetic nerves and markedly depletes NE in innervated organs. Changes in the plasma levels of NE, severity score, cytokine profiles, and percentages of immunophenotype expression in 7-day-old Bltw : CD1 (ICR) mice infected with EV71, with or without 6-OHDA treatment, were compared. The survival rate (76.9%) of EV71-infected and 6-OHDA (30 µg/g)-treated mice was increased significantly. The clinical scores were decreased markedly on days 8-12 in MP4-infected and 6-OHDA-treated mice compared to those without treatment. The results showed that the plasma levels of NE, epinephrine, and dopamine were decreased on days 4-8 after 6-OHDA treatment and at most on day 8. The plasma levels of interleukin (IL)-12p70, tumor necrosis factor, IL-6, and IL-10 did not change significantly after 6-OHDA treatment. Interferon-γ levels decreased evidently on days 4, 6, and 8 after 6-OHDA treatment. The absolute events of CD3+CD4+, CD3+CD8+, and CD3+NK1.1+ cells of peripheral blood mononuclear cells were increased significantly in MP4-infected and 6-OHDA-treated mice compared to those without treatment. In splenocytes, the absolute cells of CD3-NK1.1+, CD3+NK1.1+ and CD11b+Gr-1+ cells of EV71-infected mice were increased significantly after 6-OHDA treatment. These findings suggested that 6-OHDA may be used a probe to explore clinical improvements and immune responses in the complicated EV71 infection. Taken together, peripheral chemical sympathectomy contribute to further understand the immunopathogenesis of EV71 BE with autonomic nervous system dysregulation.

DEAD-Box Helicase DDX6 Facilitated RIG-I-Mediated Type-I Interferon Response to EV71 Infection.

Previous studies have shown that DEAD (Glu-Asp-Ala-Glu)-box RNA helicases play important roles in viral infection, either as cytosolic sensors of pathogenic molecules or as essential host factors against viral infection. In the current study, we found that DDX6, an RNA helicase belonging to the DEAD-box family of helicase, exhibited anti-Enterovirus 71 activity through augmenting RIG-I-mediated type-I IFN response. Moreover, DDX6 binds viral RNA to form an RNA-protein complex to positively regulate the RIG-I-mediated interferon response; however, EV71 has evolved a strategy to antagonize the antiviral effect of DDX6 by proteolytic degradation of the molecule through its non-structural protein 2A, a virus-encoded protease.

The expression of IFN-ß is suppressed by the viral 3D polymerase via its impact on PGAM5 expression during enterovirus D68 infection.

Enterovirus D68 (EV-D68) belongs to the Picornaviridae family and can lead to severe clinical manifestations in the respiratory system. The 3D-polymerase (3Dpoly) is an important nonstructural protein during EV-D68 replication, but few studies have addressed its interaction with the host antiviral response during EV-D68 infection. Here, we used human bronchial epithelial cells to investigate the impact of the 3Dpoly on the mitochondrial dynamics and innate immune response. The results showed that the number and morphology of the mitochondria in 16HBE cells was affected during the early stage of infection, and these effects included the cellular apoptosis. Moreover, we found that the 3Dpoly of EV-D68 can interact with PGAM5 and promote mitofusin 2 protein upregulation, and subsequently, 3Dpoly impairs IFN-ß expression by impacting the activation of the RIG-I receptor signaling pathway. Our findings suggest that during EV-D68 replication, the 3Dpoly, via its interaction with PGAM5, can affect the mitochondrial dynamics and suppress the expression of IFN-ß by impacting the RIG-I-like receptor signal pathway.

Muscle Tissue Damage and Recovery After EV71 Infection Correspond to Dynamic Macrophage Phenotypes.

Enterovirus 71 (EV71) is a positive single-stranded RNA virus from the enterovirus genus of the Picornaviridae family. Most young children infected with EV71 develop mild symptoms of hand, foot and mouth disease, but some develop severe symptoms with neurological involvement. Limb paralysis from EV71 infection is presumed to arise mainly from dysfunction of motor neurons in the spinal cord. However, EV71 also targets and damages skeletal muscle, which may also contribute to the debilitating symptoms. In this study, we have delineated the impacts of EV71 infection on skeletal muscle using a mouse model. Mouse pups infected with EV71 developed limb paralysis, starting at day 3 post-infection and peaking at day 5-7 post-infection. At later times, mice recovered gradually but not completely. Notably, severe disease was associated with high levels of myositis accompanied by muscle calcification and persistent motor end plate abnormalities. Interestingly, macrophages exhibited a dynamic change in phenotype, with inflammatory macrophages (CD45+CD11b+Ly6Chi) appearing in the early stage of infection and anti-inflammatory/restorative macrophages (CD45+CD11b+Ly6Clow/-) appearing in the late stage. The presence of inflammatory macrophages was associated with severe inflammation, while the restorative macrophages were associated with recovery. Altogether, we have demonstrated that EV71 infection causes myositis, muscle calcification and structural defects in motor end plates. Subsequent muscle regeneration is associated with a dynamic change in macrophage phenotype.

Functional and structural characterization of a two-MAb cocktail for delayed treatment of enterovirus D68 infections.

Enterovirus D68 (EV-D68) is an emerging pathogen associated with respiratory diseases and/or acute flaccid myelitis. Here, two MAbs, 2H12 and 8F12, raised against EV-D68 virus-like particle (VLP), show distinct preference in binding VLP and virion and in neutralizing different EV-D68 strains. A combination of 2H12 and 8F12 exhibits balanced and potent neutralization effects and confers broader protection in mice than single MAbs when given at onset of symptoms. Cryo-EM structures of EV-D68 virion complexed with 2H12 or 8F12 show that both antibodies bind to the canyon region of the virion, creating steric hindrance for sialic acid receptor binding. Additionally, 2H12 binding can impair virion integrity and trigger premature viral uncoating. We also capture an uncoating intermediate induced by 2H12 binding, not previously described for picornaviruses. Our study elucidates the structural basis and neutralizing mechanisms of the 2H12 and 8F12 MAbs and supports further development of the 2H12/8F12 cocktail as a broad-spectrum therapeutic agent against EV-D68 infections in humans.

MiR-103/miR-107 inhibits enterovirus 71 replication and facilitates type I interferon response by regulating SOCS3/STAT3 pathway.

BACKGROUND: Enterovirus71 (EV71), the major cause of hand, foot, and-mouth disease (HFMD), has increasingly become a public health challenge. Type I interferons (IFNs) can regulate innate and adaptive immune responses to pathogens. MicroRNAs (miRNAs) play regulatory roles in host innate immune responses to viral infections. However, the roles of miR-103 and miR-107 in EV71 infection remain unclear. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expression of miR-103, miR-107, suppressor of cytokine signaling 3 (SOCS3), VP1, IFN-α, and IFN-ß. Virus titers were measured by 50% tissue culture infectious dose (TCID50) assay. Western blot assay was conducted to detect the protein levels of VP1, IFN-α, IFN-ß, SOCS3, signal transducer and activator of transcription 3 (STAT3), and phospho-STAT3 (p-STAT3). Immunofluorescence assay was used to detect the protein level of VP1. The concentrations of IFN-α and IFN-ß were examined by Enzyme-linked immunosorbent assay (ELISA). The interaction between SOCS3 and miR-103/miR-107 was predicted by starBase and verified by dual-luciferase reporter assay and RNA pull-down assay. RESULTS: MiR-103 and miR-107 were downregulated and SOCS3 was upregulated in serum from patients with EV71 and EV71-infected cells. Overexpression of miR-103 and miR-107 repressed EV71 replication by inhibiting EV71 titers and VP1 expression. Moreover, upregulation of miR-103 and miR-107 enhanced EV71-triggered the production of type I IFNs. In addition, miR-103 and miR-107 directly targeted SOCS3, and SOCS3 upregulation reversed the effects of miR-103 and miR-107 on EV71 replication and type I IFN response. Importantly, miR-103 and miR-107 increased STAT3 phosphorylation by targeting SOCS3 after EV71 infection. CONCLUSION: MiR-103 and miR-107 suppressed EV71 replication and increased the production of type I IFNs by regulating SOCS3/STAT3 pathway, which might provide a novel strategy for developing effective antiviral therapy.

Cross-Antigenicity between EV71 Sub-Genotypes: Implications for Vaccine Efficacy.

Enterovirus A-71 (EV71) is a global, highly contagkkious pathogen responsible for severe cases of hand-food-mouth-disease (HFMD). The use of vaccines eliciting cross neutralizing antibodies (NTAbs) against the different circulating EV71 sub-genotypes is important for preventing HFMD outbreaks. Here, we tested the cross-neutralizing activities induced by EV71 genotype/sub-genotype A, B0-B4, C1, C2, C4, and C5 viruses using rats. Differences were noted in the cross-neutralization of the 10 sub-genotypes tested but there were generally good levels of cross-neutralization except against genotype A virus, against which neutralization antibody titres (NTAb) where the lowest with NTAbs being the highest against sub-genotype B4. Moreover, NTAb responses induced by C4, B4, C1, and C2 viruses were homogenous, with values of maximum/minimum NTAb ratios (MAX/MIN) against all B and C viruses ranging between 4.0 and 6.0, whereas MAX/MIN values against B3 and A viruses were highly variable, 48.0 and 256.0, respectively. We then dissected the cross-neutralizing ability of sera from infants and children and rats immunized with C4 EV71 vaccines. Cross-neutralizing titers against the 10 sub-genotypes were good in both vaccinated infants and children and rats with the MAX/MIN ranging from 1.8-3.4 and 5.1-7.1, respectively, which were similar to those found in naturally infected patients (2.8). Therefore, we conclude that C4 EV71 vaccines can provide global protection to infants and children against HFMD caused by different sub-genotypes.