RESUMEN
Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (≥99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.
Asunto(s)
Candida , Fotoquimioterapia , Humanos , Rosa Bengala/farmacología , Candida glabrata , Fármacos Fotosensibilizantes/farmacologíaRESUMEN
Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID50) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.
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Desoxiadenosinas , Enterovirus Humano A , Infecciones por Enterovirus , Enterovirus , Enfermedad de Boca, Mano y Pie , Animales , Chlorocebus aethiops , Lactante , Niño , Humanos , Preescolar , Enterovirus Humano A/genética , Células Vero , Adenosina/farmacología , Células CACO-2 , Replicación Viral , Infecciones por Enterovirus/tratamiento farmacológico , Antígenos Virales , Antivirales/farmacologíaRESUMEN
We characterized a family diagnosed with immunodeficiency disease presenting with low immunoglobulin levels and skin dyskeratosis. Exome sequencing revealed compound heterozygous missense variants in SLC5A6, the gene encoding a cellular sodium-dependent multivitamin transporter (SMVT) responsible for transporting vitamins, including biotin (vitamin B7). We showed that the biotin deficiency was caused by the SLC5A6 variants resulting in defective B cell differentiation and antibody deficiency. Altered cellular metabolic profiles, including aberrant mitochondrial respiration and reliance on glycolysis, may underlie the failure in plasma cell maturation. Replenishment of biotin improved plasma cell maturation and recovered the antibody producing activity in the patient and in a CRISPR-Cas9 gene-edited mouse model bearing a patient-specific SLC5A6 variant. Our results demonstrate the critical role of metabolic reprogramming in the maturation of plasma cells and nominate SLC5A6 as a causative gene for immunodeficiency that may be treated by biotin replenishment.
Asunto(s)
Biotina , Deficiencia de Biotinidasa , Animales , Humanos , Ratones , Linfocitos B/metabolismo , Biotina/metabolismo , Deficiencia de Biotinidasa/genética , MutaciónRESUMEN
Fungal keratitis is a serious clinical infection on the cornea caused by fungi and is one of the leading causes of blindness in Asian countries. The treatment options are currently limited to a few antifungal agents. With the increasing incidence of drug-resistant infections, many patients fail to respond to antibiotics. Riboflavin-mediated corneal crosslinking (similar to photodynamic therapy (PDT)) for corneal ectasia was approved in the US in the early 2000s. Current evidence suggests that PDT could have the potential to inhibit fungal biofilm formation and overcome drug resistance by using riboflavin and rose bengal as photosensitizers. However, only a few clinical trials have been initiated in anti-fungal keratitis PDT treatment. Moreover, the removal of the corneal epithelium and repeated application of riboflavin and rose bengal are required to improve drug penetration before and during PDT. Thus, an improvement in trans-corneal drug delivery is mandatory for a successful and efficient treatment. In this article, we review the studies published to date using PDT against fungal keratitis and aim to enhance the understanding and awareness of this research area. The potential of modifying photosensitizers using nanotechnology to improve the efficacy of PDT on fungal keratitis is also briefly reviewed.
RESUMEN
Human cardiac progenitor cells isolated from the same host may have advantages over other sources of stem cells. The aim of this study is to establish a new source of human progenitor cells collected from a waste product, pericardiac effusion fluid, after open-heart surgery in children with congenital heart diseases. The fluid was collected every 24 h for 2 days after surgery in 37 children. Mononuclear cells were isolated and expanded in vitro. These pericardial effusion-derived progenitor cells (PEPCs) exhibiting cardiogenic lineage markers, were highly proliferative and enhanced angiogenesis in vitro. Three weeks after stem cell transplantation into the ischemic heart in mice, cardiac ejection fraction was improved significantly without detectable progenitor cells. Gene expression profiles of the repaired hearts revealed activation of several known repair mechanisms including paracrine effects, cell migration, and angiogenesis. These progenitor cells may have the potential for heart regeneration.
RESUMEN
Enterovirus A71 (EV-A71) has emerged as a major pathogen causing hand, foot, and mouth disease, as well as neurological disorders. The host immune response affects the outcomes of EV-A71 infection, leading to either resolution or disease progression. However, the mechanisms of how the mammalian innate immune system detects EV-A71 infection to elicit antiviral immunity remain elusive. Here, we report that the Toll-like receptor 3 (TLR3) is a key viral RNA sensor for sensing EV-A71 infection to trigger antiviral immunity. Expression of TLR3 in HEK293 cells enabled the cells to sense EV-A71 infection, leading to type I, IFN-mediated antiviral immunity. Viral double-stranded RNA derived from EV-A71 infection was a key ligand for TLR3 detection. Silencing of TLR3 in mouse and human primary immune cells impaired the activation of IFN-ß upon EV-A71 infection, thus reinforcing the importance of the TLR3 pathway in defending against EV-A71 infection. Our results further demonstrated that TLR3 was a target of EV-A71 infection. EV-A71 protease 2A was implicated in the downregulation of TLR3. Together, our results not only demonstrate the importance of the TLR3 pathway in response to EV-A71 infection, but also reveal the involvement of EV-A71 protease 2A in subverting TLR3-mediated antiviral defenses.
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Cisteína Endopeptidasas/inmunología , Enterovirus Humano A/inmunología , ARN Viral/inmunología , Receptor Toll-Like 3/inmunología , Animales , Células Cultivadas , Regulación hacia Abajo , Enterovirus Humano A/enzimología , Silenciador del Gen , Células HEK293 , Humanos , Inmunidad Innata , Interferón beta/inmunología , Ratones , Ratones Endogámicos C57BL , ARN Bicatenario/inmunología , Receptor Toll-Like 3/genéticaRESUMEN
BACKGROUND: Enterovirus 71 (EV71) infections may be associated with neurological complications, including brainstem encephalitis (BE). Severe EV71 BE may be complicated with autonomic nervous system (ANS) dysregulation and/or pulmonary edema (PE). ANS dysregulation is related to the overactivation of the sympathetic nervous system, which results from catecholamine release. OBJECTIVE: The aims of this study were to explore the effects of catecholamines on severe EV71 infection and to investigate the changes in the percentages of EV71-infected cells, virus titer, and cytokine production on the involvement of catecholamines. STUDY DESIGN: Plasma levels of norepinephrine (NE) and epinephrine (EP) in EV71-infected patients were measured using an enzyme-linked immunoassay. The expression of adrenergic receptors (ADRs) on RD, A549, SK-N-SH, THP-1, Jurkat and human peripheral blood mononuclear cells (hPBMCs) were detected using flow cytometry. The percentages of EV71-infected cells, virus titer, and cytokine production were investigated after treatment with NE and EP. RESULTS: The plasma levels of NE and EP were significantly higher in EV71-infected patients with ANS dysregulation and PE than in controls. Both α1A- and ß2-ADRs were expressed on A549, RD, SK-N-SH, HL-60, THP-1, Jurkat cells and hPBMCs. NE treatment elevated the percentages of EV71-infected cells to 62.9% and 22.7% in THP-1 and Jurkat cells, respectively. Via treatment with EP, the percentages of EV71-infected cells were increased to 64.6% and 26.9% in THP-1 and Jurkat cells. The percentage of EV71-infected cells increased upon NE or EP treatment while the α- and ß-blockers reduced the percentages of EV71-infected cells with NE or EP treatment. At least two-fold increase in virus titer was observed in EV71-infected A549, SK-N-SH and hPBMCs after treatment with NE or EP. IL-6 production was enhanced in EV71-infected hPBMCs at a concentration of 102 pg/mL NE. CONCLUSION: The plasma levels of NE and EP elevated in EV71-infected patients with ANS dysregulation and PE. Both NE and EP enhanced the percentages of infected cells and virus titers in EV71 infection in vitro. NE and EP may play a role in the pathogenesis of EV71 BE complicated with ANS dysregulation and PE.
Asunto(s)
Infecciones por Enterovirus/virología , Enterovirus/patogenicidad , Epinefrina/sangre , Norepinefrina/sangre , Sistema Nervioso Autónomo/efectos de los fármacos , Sistema Nervioso Autónomo/virología , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/virología , Línea Celular , Línea Celular Tumoral , Preescolar , Citocinas/metabolismo , Encefalitis/sangre , Encefalitis/tratamiento farmacológico , Encefalitis/virología , Infecciones por Enterovirus/sangre , Infecciones por Enterovirus/tratamiento farmacológico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lactante , Recién Nacido , Células Jurkat , Leucocitos Mononucleares/citología , Masculino , Edema Pulmonar/tratamiento farmacológico , Edema Pulmonar/virologíaRESUMEN
UNLABELLED: Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible mechanisms, including cytokine storm, brain stem encephalitis, and fulminant pulmonary edema. Gamma interferon (IFN-γ) may confer anti-EV71 activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. This study, investigating the role of IFN-γ in EV71 infection using a murine model, showed that IFN-γ was elevated. Moreover, IFN-γ receptor-deficient mice showed higher mortality rates and more severe disease progression with slower viral clearance than wild-type mice. In vitro results showed that IFN-γ pretreatment reduced EV71 yield, whereas EV71 infection caused IFN-γ resistance with attenuated IFN-γ signaling in IFN regulatory factor 1 (IRF1) gene transactivation. To study the immunoediting ability of EV71 proteins in IFN-γ signaling, 11 viral proteins were stably expressed in cells without cytotoxicity; however, viral proteins 2A and 3D blocked IFN-γ-induced IRF1 transactivation following a loss of signal transducer and activator of transcription 1 (STAT1) nuclear translocation. Viral 3D attenuated IFN-γ signaling accompanied by a STAT1 decrease without interfering with IFN-γ receptor expression. Restoration of STAT1 or blocking 3D activity was able to rescue IFN-γ signaling. Interestingly, viral 2A attenuated IFN-γ signaling using another mechanism by reducing the serine phosphorylation of STAT1 following the inactivation of extracellular signal-regulated kinase without affecting STAT1 expression. These results demonstrate the anti-EV71 ability of IFN-γ and the immunoediting ability by EV71 2A and 3D, which attenuate IFN-γ signaling through different mechanisms. IMPORTANCE: Immunosurveillance by gamma interferon (IFN-γ) may confer anti-enterovirus 71 (anti-EV71) activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. IFN-γ receptor-deficient mice showed higher mortality and more severe disease progression, indicating the anti-EV71 property of IFN-γ. However, EV71 infection caused cellular insusceptibility in response to IFN-γ stimulation. We used an in vitro system with viral protein expression to explore the novel IFN-γ inhibitory properties of the EV71 2A and 3D proteins through the different mechanisms. According to this study, targeting either 2A or 3D pharmacologically and/or genetically may sustain a cellular susceptibility in response to IFN-γ, particularly for IFN-γ-mediated anti-EV71 activity.
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Enterovirus Humano A/inmunología , Infecciones por Enterovirus/inmunología , Interacciones Huésped-Patógeno , Interferón gamma/antagonistas & inhibidores , Transducción de Señal , Proteínas Virales/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Humanos , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children's health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells. Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.
Asunto(s)
Enterovirus Humano A/metabolismo , Infecciones por Enterovirus/sangre , Galectina 1/metabolismo , Virión/metabolismo , Replicación Viral/fisiología , Niño , Enterovirus Humano A/patogenicidad , Infecciones por Enterovirus/virología , Galectina 1/sangre , Galectina 1/genética , HumanosRESUMEN
Human enterovirus 71 (EV71) has emerged as a neuroinvasive virus that is responsible for several outbreaks in the Asia-Pacific region over the past 15 years. Appropriate animal models are needed to understand EV71 neuropathogenesis better and to facilitate the development of effective vaccines and drugs. Non-human primate models have been used to characterize and evaluate the neurovirulence of EV71 after the early outbreaks in late 1990s. However, these models were not suitable for assessing the neurovirulence level of the virus and were associated with ethical and economic difficulties in terms of broad application. Several strategies have been applied to develop mouse models of EV71 infection, including strategies that employ virus adaption and immunodeficient hosts. Although these mouse models do not closely mimic human disease, they have been applied to determine the pathogenesis of and treatment and prevention of the disease. EV71 receptor-transgenic mouse models have recently been developed and have significantly advanced our understanding of the biological features of the virus and the host-parasite interactions. Overall, each of these models has advantages and disadvantages, and these models are differentially suited for studies of EV71 pathogenesis and/or the pre-clinical testing of antiviral drugs and vaccines. In this paper, we review the characteristics, applications and limitation of these EV71 animal models, including non-human primate and mouse models.
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Antivirales/administración & dosificación , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/genética , Infecciones por Enterovirus/genética , Animales , Anticuerpos Antivirales/administración & dosificación , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Enterovirus Humano A/patogenicidad , Infecciones por Enterovirus/tratamiento farmacológico , Infecciones por Enterovirus/patología , Interacciones Huésped-Parásitos/genética , Humanos , Ratones , Replicación Viral/efectos de los fármacosRESUMEN
Although self-renewal ability of adult mammalian heart has been reported, few pharmacological treatments are known to promote cardiomyocyte regeneration after injury. In this study, we demonstrate that the critical period of stem/progenitor cell-mediated cardiomyocyte replenishment is initiated within 7 days and saturates on day 10 post-infarction. Moreover, blocking the inflammatory reaction with COX-2 inhibitors may also reduce the capability of endogenous stem/progenitor cells to repopulate lost cells. Injection of the COX-2 product PGE2 enhances cardiomyocyte replenishment in young mice and recovers cell renewal through attenuating TGF-ß1 signaling in aged mice. Further analyses suggest that cardiac stem cells are PGE2-responsive and that PGE2 may regulate stem cell activity directly through the EP2 receptor or indirectly by modulating its micro-environment in vivo. Our findings provide evidence that PGE2 holds great potential for cardiac regeneration.
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Infarto del Miocardio/metabolismo , Miocitos Cardíacos/citología , Prostaglandinas E/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Corazón/fisiopatología , Humanos , Ratones , Ratones Transgénicos , Infarto del Miocardio/genética , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Regeneración , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
From March through December 2010, the incidence of vaginal septa in our SPF breeding colony of BALB/cByJNarl mice was 14.2%. In general, septa obstructed half of the vaginal orifice. Here we sought to determine the effect of this defect by comparing the reproductive performance of affected (septate) mice with that of unaffected (nonseptate) mice. Our results showed that the rates of both copulatory plugs and pregnancy were significantly lower in septate mice than in nonseptate mice. Specifically, 23 of 45 bred septate female mice (51%) had vaginal plugs compared with 49 of 68 bred nonseptate females (72%). Only 12 septate female mice (27%) had successful pregnancies, compared with 37 nonseptate females (54%). Septate mice had a 1-logfold fewer intrauterine sperm after mating than did nonseptate mice. Three cases of dystocia were noted among septate mice whereas none occurred in nonseptate mice. Septate dams had a higher percentage of septate pups (15.5%) than did nonseptate dams (6.1%). Our findings indicate that vaginal septa affect the reproductive performance of laboratory mice and that such a defect should be considered as an exclusion criterion for the selection of future breeders in a mouse colony.
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Infertilidad Femenina/veterinaria , Ratones Endogámicos BALB C/fisiología , Reproducción/fisiología , Enfermedades de los Roedores/patología , Vagina/anomalías , Animales , Cruzamiento , Femenino , Incidencia , Infertilidad Femenina/epidemiología , Infertilidad Femenina/patología , Masculino , Ratones , Ratones Endogámicos , Embarazo , Enfermedades de los Roedores/epidemiología , Enfermedades de los Roedores/fisiopatología , Espermatozoides/citología , Útero/fisiología , Vagina/fisiologíaRESUMEN
Coxsackievirus B (CVB) and enterovirus 71 (EV71) are important causes of severe enteroviral diseases in neonates or young children in Taiwan. CVB can cause fulminant hepatitis, myocarditis or meningoencephalitis. This study was designed to explore the role of coxsackievirus-adenovirus receptor (CAR) in the pathogenesis of CVB3-infected hepatocytes via in vitro and mice studies. CVB3 (CVB3/2630) was isolated from liver tissue of a neonate with fulminant hepatitis. Cell lines A549, HeLa, HEp2 and Huh-7 were maintained in Dulbecco's modified Eagle's medium. Mice progeny 1 or 7 days old were used in the experiments. Viremia was noted in 7-day-old ICR mice 2 h after intraperitoneal injection. The highest viral titers were detected in blood, liver and spleen. Histopathological studies of the liver demonstrated polymorphonuclear cell infiltration, massive hepatic cell necrosis and apoptosis. CAR was expressed more in liver than in other tissues. Expression of CAR decreased with mouse age. Anti-CAR monoclonal antibody prevented infection of Huh-7 cells from CVB3. Furthermore, anti-CAR monoclonal antibody pretreatment can reduce mortality and decrease the level of liver enzymes in CVB3-infected mice. These findings indicate that CAR plays an important role in the initiation of CVB infections and is closely associated with hepatotropism and age-specific susceptibility.
Asunto(s)
Infecciones por Coxsackievirus/patología , Enterovirus Humano B/fisiología , Enterovirus Humano B/patogenicidad , Hígado/patología , Hígado/virología , Receptores Virales/metabolismo , Tropismo Viral , Animales , Sangre/virología , Línea Celular , Infecciones por Coxsackievirus/virología , Modelos Animales de Enfermedad , Enterovirus Humano B/aislamiento & purificación , Perfilación de la Expresión Génica , Histocitoquímica , Humanos , Ratones , Ratones Endogámicos ICR , Receptores Virales/genética , Bazo/virología , Taiwán , Carga ViralRESUMEN
Antibody-dependent enhancement (ADE) of virus infections can be induced by subneutralizing concentrations of specific antibodies. We recently demonstrated ADE in human monocytes infected with enterovirus 71 (EV71). The current study was designed to extend these observations by determining the effect of ADE on the pathogenesis of EV71 infection in newborn mice. We compared the clinical manifestations, mortality, virus titer, histopathology, and serum levels of cytokines and chemokines in newborn mice pretreated with subneutralizing antibodies to EV71 or normal mouse IgG with and without virus. Seven-day-old ICR mice were pretreated with a wide range of mouse anti-EV71 IgG 24 h prior to intraperitoneal injection of EV71. Mice were protected from infection by neutralizing doses of anti-EV71 IgG ranging from 6.43 × 10⻹ to 329.6 µg/ml. Subneutralizing doses ranging from 2.01 × 10⻲ to 3.21 × 10⻹ µg/ml were found to significantly increase 14-day mortality compared to virus alone. The ADE effect was not evident at lower doses. Histopathological examination of mice given a subneutralizing dose of 8.04 × 10⻲ µg/ml revealed extensive neuronal and muscular damage compared to untreated infected controls. Higher serum levels of interferon (IFN)-γ and monocyte chemoattractant protein (MCP)-1 were noted in mice pretreated with subneutralizing doses than untreated infected controls. These findings support the concept that subneutralizing antibodies directed enhance EV71 induce ADE in newborn mice.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Acrecentamiento Dependiente de Anticuerpo , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/patología , Animales , Animales Recién Nacidos , Tronco Encefálico/patología , Citocinas/sangre , Modelos Animales de Enfermedad , Infecciones por Enterovirus/mortalidad , Femenino , Histocitoquímica , Ratones , Ratones Endogámicos ICR , Músculos/patología , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , Carga ViralRESUMEN
Type I interferons (IFNs) represent an essential innate defense mechanism for controlling enterovirus 71 (EV 71) infection. Mice inoculated with EV 71 produced a significantly lower amount of type I IFNs than those inoculated with poly (I:C), adenovirus type V, or coxsackievirus B3 (CB3). EV 71 infection, however, mounted a proinflammatory response with a significant increase in the levels of serum and brain interleukin (IL)-6, monocyte chemoattractant protein-1, tumor necrosis factor, and IFN-γ. EV 71 infection abolished both poly (I:C)- and CB3-induced type I IFN production of mice. Such effect was not extended to other enteroviruses including coxsackievirus A24, B2, B3, and echovirus 9, as mice infected with these viruses retained type I IFN responsiveness upon poly (I:C) challenge. In addition, EV 71-infected RAW264.7 cells produced significantly lower amount of type I IFNs than non-infected cells upon poly (I:C) stimulation. The inhibitory effect of EV 71 on type I IFN production was attributed to the viral protein 3C, which was confirmed using over-expression systems in both mice and RAW264.7 cells. The 3C over-expression, however, did not interfere with poly (I:C)-induced proinflammatory cytokine production. These findings indicate that EV 71 can hamper the host innate defense by blocking selectively type I IFN synthesis through the 3C viral protein.
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Cisteína Endopeptidasas/metabolismo , Enterovirus Humano A/inmunología , Enterovirus Humano A/patogenicidad , Interferón Tipo I/antagonistas & inhibidores , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Proteasas Virales 3C , Animales , Línea Celular , Evasión Inmune , Tolerancia Inmunológica , Macrófagos/inmunología , Macrófagos/virología , RatonesRESUMEN
BACKGROUND: Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD), and infection of EV71 to central nerve system (CNS) may result in a high mortality in children less than 2 years old. Although there are two highly glycosylated membrane proteins, SCARB2 and PSGL-1, which have been identified as the cellular and functional receptors of EV71, the role of glycosylation in EV71 infection is still unclear. RESULTS: We demonstrated that the attachment of EV71 to RD and SK-N-SH cells was diminished after the removal of cell surface sialic acids by neuraminidase. Sialic acid specific lectins, Maackia amurensis (MAA) and Sambucus Nigra (SNA), could compete with EV71 and restrained the binding of EV71 significantly. Preincubation of RD cells with fetuin also reduced the binding of EV71. In addition, we found that SCARB2 was a sialylated glycoprotein and interaction between SCARB2 and EV71 was retarded after desialylation. CONCLUSIONS: In this study, we demonstrated that cell surface sialic acids assist in the attachment of EV71 to host cells. Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71 to RD and SK-N-SH cells.
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Enterovirus Humano A/fisiología , Receptores Virales/metabolismo , Ácidos Siálicos/metabolismo , Acoplamiento Viral , Antivirales/metabolismo , Línea Celular Tumoral , Glicosilación , Humanos , Lectinas/metabolismo , Proteínas de Membrana de los Lisosomas/química , Proteínas de Membrana de los Lisosomas/metabolismo , Neuraminidasa/metabolismo , Receptores Depuradores/química , Receptores Depuradores/metabolismoRESUMEN
Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease. EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis. We demonstrated that mouse-adapted strain increases infectivity, resulting in higher cytotoxicity of neuron cells and mortality to neonatal mice than a non-adapted strain. Results pointed to EV71 capsid region determining viral infectivity and mouse lethality. Mutant virus with lysine to methionine substitution at VP2(149) (VP2(149M)) or glutamine to glutamic acid substitution at VP1(145) (VP1(145E)) showed greater viral titers and apoptosis. Synergistic effect of VP2(149M) and VP1(145E) double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. The dual substitution mutants markedly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2(149M) and VP1(145E) mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to viral infectivity in vitro and mouse lethality in vivo.
Asunto(s)
Proteínas de la Cápside/genética , Enterovirus Humano A/patogenicidad , Infecciones por Enterovirus/virología , Enfermedad de Boca, Mano y Pie/virología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Apoptosis , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Chlorocebus aethiops , Enterovirus Humano A/genética , Enterovirus Humano A/inmunología , Enterovirus Humano A/metabolismo , Infecciones por Enterovirus/patología , Variación Genética , Enfermedad de Boca, Mano y Pie/genética , Ratones , Proteínas Mutantes/metabolismo , Mutación , ARN Viral/biosíntesis , Células Vero , Factores de Virulencia , Acoplamiento ViralRESUMEN
BACKGROUND: Enterovirus 71 (EV71) has emerged as a neuroinvasive virus responsible for several large outbreaks in the Asia-Pacific region while virulence determinant remains unexplored. PRINCIPAL FINDINGS: In this report, we investigated increased virulence of unadapted EV71 clinical isolate 237 as compared with isolate 4643 in mice. A fragment 12 nucleotides in length in stem loop (SL) II of 237 5'-untranslated region (UTR) visibly reduced survival time and rate in mice was identified by constructing a series of infectious clones harboring chimeric 5'-UTR. In cells transfected with bicistronic plasmids, and replicon RNAs, the 12-nt fragment of isolate 237 enhanced translational activities and accelerated replication of subgenomic EV71. Finally, single nucleotide change from cytosine to uridine at base 158 in this short fragment of 5'-UTR was proven to reduce viral translation and EV71 virulence in mice. Results collectively indicated a pivotal role of novel virulence determinant C158 on virus translation in vitro and EV71 virulence in vivo. CONCLUSIONS: These results presented the first reported virulence determinant in EV71 5'-UTR and first position discovered from unadapted isolates.
Asunto(s)
Regiones no Traducidas 5'/genética , Encéfalo/virología , Infecciones por Enterovirus/genética , Enterovirus/genética , Enterovirus/patogenicidad , Secuencias Reguladoras de Ácidos Nucleicos/genética , Virulencia , Replicación Viral , Animales , Secuencia de Bases , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Enterovirus/metabolismo , Infecciones por Enterovirus/mortalidad , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Viral/genética , Homología de Secuencia de Ácido Nucleico , Tasa de SupervivenciaRESUMEN
BACKGROUND: Neonatal mice developed neurological disease and pulmonary dysfunction after an infection with a mouse-adapted human Enterovirus 71 (EV71) strain MP4. However, the hallmark of severe human EV71 infection, pulmonary edema (PE), was not evident. METHODS: To test whether EV71-induced PE required a proinflammatory cytokine response, exogenous pro-inflammatory cytokines were administered to EV71-infected mice during the late stage of infection. RESULTS: After intracranial infection of EV71/MP4, 7-day-old mice developed hind-limb paralysis, pulmonary dysfunction, and emphysema. A transient increase was observed in serum IL-6, IL-10, IL-13, and IFN-γ, but not noradrenaline. At day 3 post infection, treatment with IL-6, IL-13, and IFN-γ provoked mild PE and severe emphysema that were accompanied by pulmonary dysfunction in EV71-infected, but not herpes simplex virus-1 (HSV-1)-infected control mice. Adult mice did not develop PE after an intracerebral microinjection of EV71 into the nucleus tractus solitarii (NTS). While viral antigen accumulated in the ventral medulla and the NTS of intracerebrally injected mice, neuronal loss was observed in the ventral medulla only. CONCLUSIONS: Exogenous IL-6, IL-13, and IFN-γ treatment could induce mild PE and exacerbate pulmonary abnormality of EV71-infected mice. However, other factors such as over-activation of the sympathetic nervous system may also be required for the development of classic PE symptoms.
Asunto(s)
Enterovirus Humano A , Infecciones por Enterovirus/patología , Interferón gamma/toxicidad , Interleucina-13/toxicidad , Interleucina-6/toxicidad , Pulmón/patología , Edema Pulmonar/patología , Enfisema Pulmonar/patología , Animales , Animales Recién Nacidos , Chlorocebus aethiops , Enterovirus Humano A/inmunología , Enterovirus Humano A/patogenicidad , Infecciones por Enterovirus/sangre , Infecciones por Enterovirus/inmunología , Humanos , Interferón gamma/sangre , Interleucina-13/sangre , Interleucina-6/sangre , Pulmón/inmunología , Pulmón/virología , Ratones , Ratones Endogámicos ICR , Parálisis/inmunología , Parálisis/patología , Parálisis/virología , Edema Pulmonar/sangre , Edema Pulmonar/inmunología , Enfisema Pulmonar/inmunología , Enfisema Pulmonar/virología , Células VeroRESUMEN
In this study we tested the effectiveness of a formaldehyde-inactivated EV71 vaccine and its compatibility for co-immunization with a pentavalent vaccine that contained inactivated poliovirus (PV) vaccine. The inactivated EV71 vaccine (C2 genogroup) elicited an antibody response which broadly neutralized homologous and heterologous genogroups, including B4, C4, and B5. Pups from vaccinated dams were resistant to the EV71 challenge and had a high survival rate and a low tissue viral burden when compared to those from non-vaccinated counterparts. Co-immunization with pentavalent and inactivated EV71 vaccines elicited antibodies against the major components of the pentavalent vaccine including the PV, Bordetella pertussis, Haemophilus influenzae type b, diphtheria toxoid, and tetanus toxoid at the same levels as in mice immunized with pentavalent vaccine alone. Likewise, EV71 neutralizing antibody titers were comparable between EV71-vaccinated mice and mice co-immunized with the two vaccines. These results indicate that formaldehyde-inactivated whole virus EV71 vaccine is feasible for designing multivalent vaccines.