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1.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(2): 159-164, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32220182

RESUMO

Objective: To investigate the effects of enterovirus 71 (EV71) on mitochondrial dynamics in human Glioma U251 cells. Methods: The EV71 was replicated in Vero cells and the 50% tissue culture infective dose (TCID 50) was calculated based on the Reed-Muench formula. After the U251 cells were infected with EV71, the cellular morphology was assessed through the light microscope. The mitochondrial morphology was detected by MitoTracker Deep Red staining under laser confocal microscopy and the mitochondrial ultrastructure was visualized by transmission electron microscopy. The expressions of mitochondrial fission proteins Drp1, p-Drp1 and fusion protein Opa1 were examined by Western blot. The level of ATP was measured by a commercial ATP assay kit. The generation of mitochondrial superoxide was detected by MitoSOX staining. Results: The TCID 50 of EV71 was 10 -5.4/0.1 mL. Twenty-four or 48 h after EV71 infection, the U251 cells appeared shrunken, round and dead. The laser confocal microscopy and transmission electron microscopy images showed that the EV71 infection induced mitochondrial elongation and cristae damage. Moreover, Western blot analysis demonstrated that the protein expressions of Drp1 and Opa1 were downregulated at both 24 and 48 h after EV71 infection in U251 cells, companied with a significant increase in Drp1 phosphorylation at 48 h after infection ( P<0.05). In addition, a decreased ATP level and elevated mitochondrial superoxide generation were observed in the EV71 infected group, as compared to the control group. Conclusion: Our study demonstrated that infection with EV71 led to changes of mitochondrial morphology and dynamics in U251 cells, which may impair mitochondrial function and contribute to nervous system dysfunction.


Assuntos
Neoplasias Encefálicas/virologia , Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Glioma/virologia , Dinâmica Mitocondrial , Animais , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/complicações , Humanos , Sistema Nervoso/fisiopatologia , Sistema Nervoso/virologia , Células Tumorais Cultivadas , Células Vero
2.
Emerg Microbes Infect ; 9(1): 427-438, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32079505

RESUMO

Enterovirus 71 (EV71) is a major pathogen that causes hand, foot and mouth disease (HFMD), which is a life threatening disease in certain children. The pathogenesis of EV71-caused HFMD is poorly defined due to the lack of simple and robust animal models with severe phenotypes that recapitulate symptoms observed in humans. Here, we generated the infectious clone of a clinical isolate from a severe HFMD patient. Virus rescued from the cDNA clone was infectious in cell lines. When administrated intraperitoneally to neonatal ICR, BALB/c and C57 immune competent mice at a dosage of1.4 × 104 pfu per mouse, the virus caused weight loss, paralysis and death in the infected mice after 4-5 days of infection. In the infected mice, detectable viral replication was detected in various tissues such as heart, liver, brain, lung, kidney, small intestine, leg skeletal muscle and medulla oblongata. The histology of the infected mice included massive myolysis, glomerular atrophy, villous blunting in small intestine, widened alveolar septum, diminished alveolar spaces and lymphocytes infiltration into the lung. By using the UV-inactivated virus as a control, we elucidated that the virus first amplified in the leg skeletal muscle tissue and the muscle tissue served as a primary viral replication site. In summary, we generated a stable EV71 infectious clone that is capable of infecting neonatal immune competent mice without adaptive mutations and provide a simple, valuable animal model for the studies of EV71pathogenesis and therapy.


Assuntos
Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Doença de Mão, Pé e Boca/virologia , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , DNA Complementar , Modelos Animais de Doenças , Humanos , Lactente , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Mutação , RNA Viral , Organismos Livres de Patógenos Específicos , Células Vero , Virulência , Replicação Viral
3.
J Biomed Sci ; 26(1): 81, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31630680

RESUMO

As a neurotropic virus, enterovirus A71 (EV-A71) emerge and remerge in the Asia-Pacific region since the 1990s, and has continuously been a threat to global public health, especially in children. Annually, EV-A71 results in hand-foot-and-mouth disease (HFMD) and occasionally causes severe neurological disease. Here we reviewed the global epidemiology and genotypic evolution of EV-A71 since 1997. The natural selection, mutation and recombination events observed in the genetic evolution were described. In addition, we have updated the antigenicity and virulence determinants that are known to date. Understanding EV-A71 epidemiology, genetic evolution, antigenicity, and virulence determinants can expand our insights of EV-A71 pathogenesis, which may benefit us in the future.


Assuntos
Enterovirus Humano A , Infecções por Enterovirus/epidemiologia , Evolução Molecular , Doença de Mão, Pé e Boca/epidemiologia , Antígenos Virais/imunologia , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Genótipo , Doença de Mão, Pé e Boca/virologia , Humanos , Virulência
4.
Emerg Microbes Infect ; 8(1): 1076-1085, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339457

RESUMO

Enterovirus A71 (EV-A71) is one of the main causative agents of hand-foot-and-mouth disease and is occasionally associated with severe neurological complications. EV-A71 pathophysiology is poorly understood due to the lack of small animal models that robustly support viral replication in relevant organs/tissues. Here, we show that adult severe combined immune-deficient (SCID) mice can serve as an EV-A71 infection model to study neurotropic determinants and viral tropism. Mice inoculated intraperitoneally with an EV-A71 clinical isolate had an initial infection of the lung compartment, followed by neuroinvasion and infection of (motor)neurons, resulting in slowly progressing paralysis of the limbs. We identified a substitution (V135I) in the capsid protein VP2 as a key requirement for neurotropism. This substitution was also present in a mouse-adapted variant, obtained by passaging the clinical isolate in the brain of one-day-old mice, and induced exclusive neuropathology and rapid paralysis, confirming its role in neurotropism. Finally, we showed that this residue enhances the capacity of EV-A71 to use mouse PSGL1 for viral entry. Our data reveal that EV-A71 initially disseminates to the lung and identify viral and host determinants that define the neurotropic character of EV-A71, pointing to a hitherto understudied role of PSGL1 in EV-A71 tropism and neuropathology.


Assuntos
Proteínas do Capsídeo/genética , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Glicoproteínas de Membrana/metabolismo , Neurônios/virologia , Animais , Proteínas do Capsídeo/metabolismo , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/genética , Infecções por Enterovirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos SCID , Mutação de Sentido Incorreto , Neurônios/metabolismo , Tropismo Viral , Virulência , Internalização do Vírus
5.
J Mol Neurosci ; 69(2): 188-196, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31201655

RESUMO

Mutations of the CPT2 gene cause CPT2 deficiency and affect the ß-oxidation of fatty acids. This study examined the consequence of a polymorphism of rs1799822 in the CPT2 gene with respect to EV71 encephalitis in Chinese children. The study included 406 cases of both mild and severe EV71 infection diagnosed by RT-PCR, together with controls (n = 348). We used an improved multiplex ligation detection reaction technique to detect the polymorphism of rs1799822 in the CPT2 gene. The frequency of the (AG+GG) genotype and G allele in the EV71 infection group and in the severe EV71 encephalitis group was significantly lower than in the control group (p = 0.012 vs. p = 0.005, and p = 0.022 vs. p = 0.006, respectively). The frequency of the (AG+GG) genotype and G allele in the severe EV71 encephalitis group was markedly lower than in the mild EV71 encephalitis group (p = 0.045, p = 0.033). The ATP levels in the blood of the (AG+GG) genotype were distinctly higher than in the AA genotype in mild and severe EV71 encephalitis patients (P = 0.037, P = 0.040). A polymorphism of rs1799822 in the CPT2 gene is associated with the severity of EV71 encephalitis and may be one of the protection factors of severe EV71 encephalitis.


Assuntos
Carnitina O-Palmitoiltransferase/genética , Encefalite Viral/genética , Infecções por Enterovirus/genética , Polimorfismo de Nucleotídeo Único , Trifosfato de Adenosina/sangue , Criança , Pré-Escolar , China , Encefalite Viral/sangue , Encefalite Viral/virologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/sangue , Infecções por Enterovirus/virologia , Feminino , Humanos , Lactente , Masculino
6.
Microb Pathog ; 132: 215-221, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31075431

RESUMO

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are common causative agents of mild and self-limiting symptoms of childhood hand, foot, and mouth disease (HFMD). However, some EV71-infected HFMD patients can develop severe neurological and/or fatal cardiopulmonary complications. In Thailand, HFMD associated with the EV71 subgenotypes C4a and B5 were reported to be associated with diverse outcomes. However, variations in enterovirus subgenotypes and virulence factors have not been fully elucidated; this study elucidated these variations in peripheral blood mononuclear cells (PBMCs) exposed to different subgenotypes of isolated enteroviruses for 24 and 48 h. Following infection, viral titers were determined by plaque assay. Infected cells and intracellular cytokines were quantified using flow cytometry, and multiplex assay was used to examine cytokine release. All isolated subgenotypes showed replication capability in PBMCs; specifically, the replication titer of EV71 C4a tended to be higher than titers of EV71 B5 and CA16. Additionally, the infectivity of EV71 B5 was higher in monocytes than in lymphocytes. Compared with EV71 B5, EV71 C4a and CA16 had greater ability to induce intra- and extracellular cytokine responses. These findings provide new insights into variations in cellular immune responses to different EV71 subgenotypes isolated from Thai patients, which should be considered for the development of vaccines and therapeutic agents.


Assuntos
Citocinas/metabolismo , Enterovirus Humano A/imunologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/imunologia , Adulto , Animais , Anticorpos Monoclonais , Quimopapaína/metabolismo , Enterovirus/imunologia , Enterovirus/isolamento & purificação , Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/virologia , Feminino , Humanos , Imunidade Celular/imunologia , Leucócitos Mononucleares , Masculino , Tailândia , Células Vero , Virulência , Adulto Jovem
7.
Virus Res ; 265: 104-114, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30910697

RESUMO

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease and severe neural complications in infants and young children. Viral pathogenesis is associated with virus-induced cell death and inflammatory cytokine production, which is usually correlated with the type of programmed cell death. EV71-infected cells were analyzed through microscopy, cell staining, and immunoblotting to determine the characteristics of EV71-induced cell death. Results demonstrated that EV71 infection induced cell shrinkage, nuclear condensation, decreased mitochondrial potential, and membrane phosphatidylserine translocation. Caspase-9 activation, poly (ADP-ribose) polymerase cleavage, and lactate dehydrogenase release were also observed during virus-induced cell death. The activated gasdermin D (GSDMD) and the phosphorylated mixed lineage kinase domain-like protein (p-MLKL) were not detected. These observations indicated that EV71-induced cell death was mainly executed by apoptosis through the intrinsic pathway rather than by GSDMD-mediated pyroptosis and p-MLKL-mediated necroptosis. Genome scanning analysis identified that EV71 2A, 2B, and 3C might be the determinant genes of virus-induced cell death. Further experiments showed that EV71 2A- and 3C-induced cell death exhibited dependence on their protease activities but involved different mechanisms. EV71 2A-induced cell death was correlated with the shut-off of host cap-dependent translation, whereas EV71 3C-induced cell death might not be ascribed to this mechanism. These findings would enhance our understanding of EV71 infection and viral pathogenesis, and help identify antiviral targets.


Assuntos
Apoptose/genética , Morte Celular/genética , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Genes Virais , Caspase 9/genética , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas Virais/genética
8.
Genet Test Mol Biomarkers ; 23(3): 188-196, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30807256

RESUMO

AIM: This study was conducted to determine if single nucleotide polymorphisms within the interleukin (IL)-13 (rs20541 locus), the S100B (rs9722 locus), and the toll-like receptor (TLR)-7 (rs179019 and rs3853839 loci) genes are associated with the clinical severity of disease caused by enterovirus 71 (EV71) in children suffering from hand, foot, and mouth disease (HFMD). MATERIALS AND METHODS: A total of 355 children, diagnosed with HFMD, were divided into two groups: severe (totaling 162 cases) and mild (totaling 193 cases). Three hundred healthy children were recruited as a control group. The gene polymorphisms of the rs20541 locus in the IL-13 gene; the rs9722 locus in the S100B gene; and the rs179019 and the rs3853839 loci in the TLR-7 gene were analyzed with Sanger sequencing. The expression levels of IL-13, S100B, interferon (IFN)-α, IL-6 and the relative expression level of TLR-7 were calculated for each genotype. RESULTS: This study demonstrated that the T allele at the rs9722 locus of the S100B gene was a significant risk factor for severe HFMD. The rs3853839 C allele of the TLR-7 gene was also a risk factor for severe HFMD in both male and female patients. The G allele at the rs20541 locus of IL-13 gene and the A allele at the rs179019 locus of the TLR-7 gene were not risk factors for severe HFMD in either male or female patients. CONCLUSION: The T allele at the rs9722 locus of S100B gene is a risk factor for the severe HFMD caused by EV71 infection, of which the mechanism may be due to the promotion of S100B protein secretion. The allele C at TLR-7 rs3853839 locus is a risk factor for the severe HFMD caused by EV71 infection, which may be related to a reduction of the relative expression of TLR-7, IFN-α, and IL-6.


Assuntos
Interleucina-13/genética , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Receptor 7 Toll-Like/genética , Alelos , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , Pré-Escolar , China , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Genótipo , Doença de Mão, Pé e Boca/complicações , Doença de Mão, Pé e Boca/genética , Humanos , Lactente , Recém-Nascido , Interleucina-13/fisiologia , Masculino , Razão de Chances , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Subunidade beta da Proteína Ligante de Cálcio S100/fisiologia , Receptor 7 Toll-Like/fisiologia
9.
Virus Res ; 263: 55-63, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30611822

RESUMO

Enterovirus A71 (EV-A71) is known for its manifestation as hand foot and mouth disease (HFMD), which has caused countless large-scale epidemic outbreaks throughout the world. However, the molecular pathogenesis of EV-A71 infection is still elusive. Previous studies found that the biological characteristics of a mild EV-A71 strain (SDLY1) and a severe EV-A71 strain (SDLY107) are significantly different, and sequence analysis showed that there are several differences in nucleotide sites of UTRs (88 nt, 123 nt, 143 nt, 154 nt, 187 nt, 241 nt, 243 nt, 253 nt, 291 nt, 438 nt, 440 nt, 571 nt, 579 nt, 602 nt, 658 nt, 664 nt, 690 nt, 696 nt, 7328 nt, 7335 nt, 7367 nt, and 7395 nt). The aim of this study was to determine whether these amino sites in UTRs are associated with the pathogenesis of EV-A71 and are responsible for different clinical manifestations. Based on the reverse genetics technology, we rescued two chimeric viruses SDLY107(1-5'UTR) and SDLY107(1-3'UTR) by replacing 5'UTR/3'UTR gene fragments of an infectious cDNA clone. Replication kinetics and cytotoxicity assays showed that the virulence of the two chimeric strains significantly changed in vitro. The viral loads of the two chimeric strains in infected ICR mice were reduced and pathological damage in the brains, lungs, intestinal tissues, and muscles were lightened. Our findings suggest that some nucleotide sites in UTRs may have a function in the pathogenicity and virulence of EV-A71.


Assuntos
Enterovirus Humano A/crescimento & desenvolvimento , Enterovirus Humano A/patogenicidade , Doença de Mão, Pé e Boca/patologia , Doença de Mão, Pé e Boca/virologia , RNA Viral/genética , Regiões não Traduzidas , Fatores de Virulência , Estruturas Animais/patologia , Estruturas Animais/virologia , Animais , Linhagem Celular , Sobrevivência Celular , Modelos Animais de Doenças , Enterovirus Humano A/genética , Humanos , Camundongos Endogâmicos ICR , Genética Reversa , Carga Viral , Virulência , Replicação Viral
10.
BMC Infect Dis ; 19(1): 19, 2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30616531

RESUMO

BACKGROUND: Hand, foot and mouth disease (HFMD) is an infectious disease caused by enteroviruses that has a severely impair for those high incidence countries such as China.The current study aimed to investigate the epidemic pattern of HFMD by time and region in Northwestern China. METHODS: All reported HFMD cases from 2008 to 2015 were collected from local Disease Control and Prevention.The HFMD was diagnosed in accordance with the guidebook provided by the National Health and Family Planning Commission of the People's Republic of China. RESULTS: A total of 154,869 cases of probable HFMD were reported. The overall incidence of HFMD has been increased from 91.68 per 100/000 in 2008 to 335.64 per 100/000 in 2015.The case mortality is decreased from 0.014 per100/000 to 0.011 per 100/000 during the time period. Most HFMD (93.82%) occurred in children younger than 5 years. The seasonal peak of HFMD infections occurred in April-July and September-November and Central regions of Xi'an city were the major locations of the clusters (incidence rate 245.75/100,000; relative risk 1.19, P < 0.01). EVA71 was the predominant enterovirus serotype, accounting for 50.0% of all reported HFMD cases since 2011.The most susceptible group infected by HFMD was children younger than 5 years, especially boys. CONCLUSIONS: Incidence of HFMD has been increasing in the past few years, however, the case fatality is decreasing. Season and region shall be considered as influence factors in the prevention of HFMD.


Assuntos
Doença de Mão, Pé e Boca/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Pré-Escolar , China/epidemiologia , Cidades , Enterovirus Humano A/classificação , Enterovirus Humano A/patogenicidade , Epidemias , Feminino , Doença de Mão, Pé e Boca/mortalidade , Doença de Mão, Pé e Boca/virologia , Humanos , Incidência , Lactente , Masculino , Pessoa de Meia-Idade , Mortalidade , Estações do Ano , Sorogrupo
11.
Pediatr Infect Dis J ; 38(2): 99-103, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29624567

RESUMO

BACKGROUND: Brainstem encephalitis is the most common neurologic complication after enterovirus 71 infection. The involvement of brainstem, especially the dorsal medulla oblongata, can cause severe sequelae or death in children with enterovirus 71 infection. We aimed to determine the prevalence of dorsal medulla oblongata involvement in children with enterovirus 71-related brainstem encephalitis (EBE) by using conventional magnetic resonance imaging (MRI) and to evaluate the value of dorsal medulla oblongata involvement in outcome prediction. METHODS: Forty-six children with EBE were enrolled in the study. All subjects underwent a 1.5 Tesla MRI examination of the brain. The disease distribution and clinical data were collected. Dichotomized outcomes (good vs. poor) at longer than 6 months were available for 28 patients. Logistic regression was used to determine whether the MRI-confirmed dorsal medulla oblongata involvement resulted in improved clinical outcome prediction when compared with other location involvement. RESULTS: Of the 46 patients, 35 had MRI evidence of dorsal medulla oblongata involvement, 32 had pons involvement, 10 had midbrain involvement and 7 had dentate nuclei involvement. Patients with dorsal medulla oblongata involvement or multiple area involvement were significantly more often in the poor outcome group than in the good outcome group. Logistic regression analysis showed that dorsal medulla oblongata involvement was the most significant single variable in outcome prediction (predictive accuracy, 90.5%), followed by multiple area involvement, age and initial Glasgow Coma Scale score. CONCLUSIONS: Dorsal medulla oblongata involvement on conventional MRI correlated significantly with poor outcomes in EBE children, improved outcome prediction when compared with other clinical and disease location variables, and was most predictive when combined with multiple area involvement, Glasgow Coma Scale score and age.


Assuntos
Tronco Encefálico/virologia , Encefalite/diagnóstico por imagem , Infecções por Enterovirus/diagnóstico por imagem , Imagem por Ressonância Magnética , Bulbo/diagnóstico por imagem , Encefalite/virologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/complicações , Feminino , Humanos , Lactente , Modelos Logísticos , Masculino , Estudos Retrospectivos
12.
Rev Med Virol ; 29(1): e2016, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30378208

RESUMO

Enteroviruses are members of Pichornaviridae family consisting of human enterovirus group A, B, C, and D as well as nonhuman enteroviruses. Hand, foot, and mouth disease (HFMD) is a serious disease which is usually seen in the Asia-Pacific region in children. Enterovirus 71 and coxsackievirus A16 are two important viruses responsible for HFMD which are members of group A enterovirus. IFN α and ß are two cytokines, which have a major activity in the innate immune system against viral infections. Most of the viruses have some weapons against these cytokines. EV71 has two main proteases called 2A and 3C, which are important for polyprotein processing and virus maturation. Several studies have indicated that they have a significant effect on different cellular pathways such as interferon production and signaling pathway. The aim of this study was to investigate the latest findings about the interaction of 2A and 3C protease of EV71 and IFN production/signaling pathway and their inhibitory effects on this pathway.


Assuntos
Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/patogenicidade , Doença de Mão, Pé e Boca/virologia , Evasão da Resposta Imune , Fatores Imunológicos/antagonistas & inibidores , Interferon Tipo I/antagonistas & inibidores , Proteínas Virais/metabolismo , Ásia/epidemiologia , Enterovirus Humano A/enzimologia , Doença de Mão, Pé e Boca/epidemiologia , Humanos
13.
J Virol ; 93(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30429352

RESUMO

Since the discovery of enterovirus A71 (EV-A71) half a century ago, it has been recognized as the cause of large-scale outbreaks of hand-foot-and-mouth disease worldwide, particularly in the Asia-Pacific region, causing great concern for public health and economic burdens. Detailed mechanisms on the modulation of immune responses after EV-A71 infection have not been fully known, and the lack of appropriate models hinders the development of promising vaccines and drugs. In the present study, NOD-scid IL2Rγ-/- (NSG) mice with a human immune system (humanized mice) at the age of 4 weeks were found to be susceptible to a human isolate of EV-A71 infection. After infection, humanized mice displayed limb weakness, which is similar to the clinical features found in some of the EV-A71-infected patients. Histopathological examination indicated the presence of vacuolation, gliosis, or meningomyelitis in brain stem and spinal cord, which were accompanied by high viral loads detected in these organs. The numbers of activated human CD4+ and CD8+ T cells were upregulated after EV-A71 infection, and EV-A71-specific human T cell responses were found. Furthermore, the secretion of several proinflammatory cytokines, such as human gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-17A, was elevated in the EV-A71-infected humanized mice. Taken together, our results suggested that the humanized mouse model permits insights into the human immune responses and the pathogenesis of EV-A71 infection, which may provide a platform for the evaluation of anti-EV-A71 drug candidates in the future.IMPORTANCE Despite causing self-limited hand-food-and-mouth disease in younger children, EV-A71 is consistently associated with severe forms of neurological complications and pulmonary edema. Nevertheless, only limited vaccines and drugs have been developed over the years, which is possibly due to a lack of models that can more accurately recapitulate human specificity, since human is the only natural host for wild-type EV-A71 infection. Our humanized mouse model not only mimics histological symptoms in patients but also allows us to investigate the function of the human immune system during infection. It was found that human T cell responses were activated, accompanied by an increase in the production of proinflammatory cytokines in EV-A71-infected humanized mice, which might contribute to the exacerbation of disease pathogenesis. Collectively, this model allows us to delineate the modulation of human immune responses during EV-A71 infection and may provide a platform to evaluate anti-EV-A71 drug candidates in the future.


Assuntos
Linfócitos T CD8-Positivos/patologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/patologia , Feto/patologia , Carga Viral/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Feto/imunologia , Feto/virologia , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
14.
Genes Genomics ; 41(3): 343-357, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30499052

RESUMO

BACKGROUND: Enterovirus 71 (EV71) is the main pathogen of hand-foot-mouth disease (HFMD) and sometimes causes several neurological complications. However, the underlying mechanism of the host response to the virus infection remains unclear. OBJECTIVE: To reveal the cell-specific transcriptional response of cultured RD cells following infection with EV71, and better understand the molecular mechanisms of virus-host interactions. METHODS: The RD cells were infected with or without EV71 for 24 h, and then transcriptome sequencing and qRT-PCR were performed to analyze the transcriptome difference of functional genes. RESULTS: More than 15000 genes were identified in transcriptome sequencing. In comparison with uninfected RD cells, 329 DEGs were identified in cells infected with EV71. GO and KEGG pathway enrichment analysis showed that most of the DEGs were related to DNA binding, transcriptional regulation, immune response and inflammatory response, apoptosis inducing factors and enriched in JAK-STAT and MAPK signaling pathways. TXNIP (thioredoxin-interacting protein) gene was further demonstrated to play an important role participating in cellular apoptosis induced by EV71, and the apoptosis and death mediated by TXNIP during EV71 infection was triggered by viral 2A protease (2Apro), not 3C protease (3Cpro). CONCLUSION: Our study demonstrated that RD cells have a significant response to EV71 infection, including immune response and apoptosis. 2Apro might be a key inducer relative to the cellular apoptosis and death mediated by TXNIP during EV71 infection. These data would contribute to preferably understand the process at the molecular level and provide theoretical foundation for diagnosis and treatment of EV71-related diseases.


Assuntos
Apoptose , Proteínas de Transporte/genética , Cisteína Endopeptidases/genética , Infecções por Enterovirus/genética , Transcriptoma , Proteínas Virais/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/enzimologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Proteínas Virais/metabolismo
15.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30487277

RESUMO

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.IMPORTANCE Recombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.


Assuntos
Enterovirus Humano A/genética , Recombinação Genética/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Surtos de Doenças , Enterovirus/genética , Enterovirus Humano A/metabolismo , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Genoma Viral/genética , Humanos , Mutação , Filogenia , Poliomielite/epidemiologia , Poliomielite/virologia , Estudos Retrospectivos , Virulência
16.
Biomarkers ; 24(3): 277-285, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30521401

RESUMO

Purpose: Hypercatecholaminemia-related heart failure has been proposed as the main cause of enterovirus A71-related (EV-A71) early mortality. The purpose of this study was to measure urine catecholamine concentrations in severe EV-A71-infected children. Methods: A total of 35 children, aged 2.5 ± 2.1 years, were divided into three groups. Group I: 15 septic shock patients, group II: 17 EV-A71-stage-2 patients, and group III: 3 EV-A71-stage-4 patients. The laboratory results, cardiac biomarkers and urine catecholamine concentrations were statistically analysed. Results: Group I had the highest C-reactive protein (CRP) levels and group II had the lowest B-type natriuretic peptide (BNP) and its N-terminal prohormone among the groups (p = 0.039, <0.01 and <0.01, respectively). Group III patients had significantly higher urine catecholamine and troponin-I values among the groups. If urine epinephrine (Epi) >134 ug/gCr, norepinephrine (NE) >176 ug/gCr and vanillylmandelic acid (VMA) >11.7 mg/gCr were used as the cutoff points to differentiate groups II and III, the sensitivities and specificity were all 100%. Conclusions: The significantly elevated urine catecholamine concentrations in EV-A71-stage-4 patients support the hypothesis that hypercatecholaminemia-related heart failure is involved in severe EV-A71 infection. Urine catecholamines could be used as reliable biomarkers for differentiation of severe EV-A71 infection with or without heart failure and septic shock.


Assuntos
Catecolaminas/urina , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/urina , Choque Séptico/urina , Criança , Pré-Escolar , Infecções por Enterovirus/patologia , Infecções por Enterovirus/virologia , Feminino , Humanos , Lactente , Masculino , Choque Séptico/patologia , Choque Séptico/virologia
17.
Virol Sin ; 34(1): 9-21, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30552635

RESUMO

Enterovirus 71 (EV71) is one of the main pathogens that causes hand-foot-and-mouth disease (HFMD). HFMD caused by EV71 infection is mostly self-limited; however, some infections can cause severe neurological diseases, such as aseptic meningitis, brain stem encephalitis, and even death. There are still no effective clinical drugs used for the prevention and treatment of HFMD. Studying EV71 protein function is essential for elucidating the EV71 replication process and developing anti-EV71 drugs and vaccines. In this review, we summarized the recent progress in the studies of EV71 non-coding regions (5' UTR and 3' UTR) and all structural and nonstructural proteins, especially the key motifs involving in viral infection, replication, and immune regulation. This review will promote our understanding of EV71 virus replication and pathogenesis, and will facilitate the development of novel drugs or vaccines to treat EV71.


Assuntos
Enterovirus Humano A/genética , Genômica , Proteínas Virais/genética , Replicação Viral/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/complicações , Infecções por Enterovirus/virologia , Genoma Viral , Doença de Mão, Pé e Boca/virologia , Humanos , Camundongos , RNA não Traduzido/genética , Proteínas Virais/metabolismo
18.
Microb Pathog ; 128: 106-111, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30579945

RESUMO

As one of the major causative agents of hand, foot and mouth disease (HFMD), enterovirus 71 (EV71) is a small, non-enveloped positive stranded RNA virus. Children suffering EV71 infection may cause severe symptoms including neurological complications, pulmonary edema and aseptic meningitis. EV71 is a neurotropic virus and it can cause the damage of nervous cells, cytokine storm and toxic substance. Identifying the factors that mediate viral binding or entry to host cells is important to uncover the mechanisms which viruses utilize to cause diseases in human body. Heat shock protein 70 (HSP70) is induced during virus infection and facilitates proper protein folding during viral propagation. The role that HSP70 plays during EV71 infection is still unclear. In this study, siRNA interference technique and transgenic technique were used to investigate the interaction between HSP70 and EV71 virus. The result demonstrated that the cell surface HSP70 is not essential for EV71 infection but helps the initial binding of virus to host cells and that multiple receptors are involved during EV71 infection. In addition, HSP70 was upregulated in human neuroblastoma cells (SK-N-SH) infected with EV71.


Assuntos
Enterovirus Humano A/metabolismo , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Proteínas de Choque Térmico HSP70/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP70/genética , Humanos , Neuroblastoma/virologia , Neurônios/virologia , RNA Interferente Pequeno , Regulação para Cima , Ligação Viral , Internalização do Vírus , Replicação Viral/fisiologia
19.
Emerg Microbes Infect ; 7(1): 205, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518755

RESUMO

Enterovirus A71 (EV-A71) is a major etiological agent of human hand, foot and mouth disease, and it can cause severe neurological complications. Although several genotypes of EV-A71 strains are prevalent in different regions of the world, the genotype C4 has circulated in mainland China for more than 20 years. The pathogenicity of different EV-A71 clinical isolates varies and needs to be explored. In this study, hSCARB2 knock-in mice (N = 181) with a wide range of ages were tested for their susceptibility to two EV-A71 strains with the subgenotypes C4 and C2, and two infection routes (intracranial and venous) were compared. The clinical manifestations and pathology and their relationship to the measured viral loads in different tissues were monitored. We observed that 3 weeks is a crucial age, as mice younger than 3-week-old that were infected became extremely ill. However, mice older than 3 weeks displayed diverse clinical symptoms. Significant differences were observed in the pathogenicity of the two strains with respect to clinical signs, disease incidence, survival rate, and body weight change. We concluded that hSCARB2 knock-in mice are a sensitive model for investigating the clinical outcomes resulting from infection by different EV-A71 strains. The intracranial infection model appears to be suitable for evaluating EV-A71 neurovirulence, whereas the venous infection model is appropriate for studying the pathogenicity of EV-A71.


Assuntos
Encéfalo/virologia , Modelos Animais de Doenças , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Receptores Depuradores/genética , Administração Intravenosa , Fatores Etários , Animais , Antígenos Virais/genética , Enterovirus Humano A/genética , Infecções por Enterovirus/sangue , Técnicas de Introdução de Genes , Genótipo , Humanos , Camundongos , Crânio/virologia , Carga Viral , Virulência
20.
PLoS One ; 13(11): e0206769, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30395634

RESUMO

OBJECTIVE: To evaluate the association of enterovirus 71 (EV71) susceptibility and clinical severity with polymorphisms in EV71 receptors, including human scavenger receptor class B member 2 (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), and annexin II (ANXA2). METHODS: We enrolled laboratory-confirmed EV71 cases and healthy age- and gender-matched controls in Taiwan from 2000 to 2012. We detected genetic polymorphisms in SCARB2, PSGL-1, and ANXA2 and correlated the results with EV71 susceptibility and severity. RESULTS: We collected 599 EV71 cases and 98 controls. Among EV71 patients, the male to female ratio was 1.61, and the mean age was 2.99±2.47 years. For clinical severity, 117 (19.6%) had severe central nervous system involvement with or without cardiopulmonary failure. For outcomes, 46 (7.7%) had sequelae, and 14 (2.3%) died. SCARB2 polymorphisms (rs6824953 and rs11097262) were associated with susceptibility to EV71 infection (OR 1.60, 95% CI 1.07-2.39; and OR 1.64, 95% CI 1.09-2.47, respectively). PSGL-1 polymorphisms (rs7137098 and rs8179137) were significantly associated with severe EV71 infection (OR 1.46, 95% CI 1.1-1.96; and OR 1.47, 95% CI 1.07-2.03, respectively). CONCLUSIONS: SCARB2 polymorphisms (rs6824953 and rs11097262) might be associated with EV71 susceptibility. PSGL-1 polymorphisms (rs7137098 and rs8179137) were associated with severe EV71 infection.


Assuntos
Anexina A2/genética , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , Receptores Depuradores/genética , Receptores Virais/genética , Estudos de Casos e Controles , Pré-Escolar , Infecções por Enterovirus/virologia , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Índice de Gravidade de Doença , Taiwan
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