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1.
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
2.
Nucleic Acids Res ; 48(3): 1392-1405, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31863580

RESUMO

The enterovirus 71 (EV71) 3Dpol is an RNA-dependent RNA polymerase (RdRP) that plays the central role in the viral genome replication, and is an important target in antiviral studies. Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5'-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity (KM,NTP). We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.


Assuntos
Enterovirus Humano A/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , RNA Replicase/ultraestrutura , Antivirais/química , Sítios de Ligação , Cristalografia por Raios X , Enterovirus Humano A/química , Enterovirus Humano A/genética , Genoma Viral , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Nucleotídeos/química , RNA Replicase/química , RNA Viral/química , RNA Viral/ultraestrutura , Replicação Viral/genética
3.
Arch Virol ; 164(12): 2975-2984, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31570994

RESUMO

Coxsackievirus A16 (CV-A16) is one of the main causative agents of hand, foot and mouth disease (HFMD) in young children and has become prevalent in the Asia-Pacific region in recent years. However, no approved vaccines or drugs are available for CV-A16 infection. CV-A16 virus-like particles (VLPs) are a potential vaccine candidate; however, whether the intranasal route of immunization is suitable for inducing immune responses against CV-A16 infection has not been clarified. In this study, the comprehensive immunogenicity and protective efficacy of the CV-A16 VLP vaccine were evaluated by multiple methods in a mouse model. In mice, a high neutralizing antibody (NTAb) titre could be elicited by intranasal immunization with CV-A16 VLPs, which produced NTAb levels similar to those induced by intranasal immunization with inactivated CV-A16. Passive immunity with NTAbs provided very good protection, as the survival rate of the immunized neonatal mice was 100% after challenges with CV-A16 at a dose of 1000 LD50. Passive protective effects were transferred to the neonates via the mother, thus protecting all the pups against challenges with the homologous or heterologous strains of CV-A16 at a dose of 1000 LD50. In addition, intranasal immunization with CV-A16 VLPs also induced the production of mucosal secretory IgA (s-IgA) antibodies, which may inhibit CV-A16 virus invasion. This study provides valuable supplemental information to facilitate our understanding of the specific protective efficacy of CV-A16 VLPs and has significance for development of the candidate vaccine into a safe and effective vaccine.


Assuntos
Enterovirus Humano A/imunologia , Infecções por Enterovirus/prevenção & controle , Nariz/virologia , Vacinas Virais/administração & dosagem , Animais , Animais Recém-Nascidos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Enterovirus Humano A/genética , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos ICR , Vacinas Virais/genética , Vacinas Virais/imunologia
4.
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
5.
J Biomed Sci ; 26(1): 75, 2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31627753

RESUMO

Enterovirus A71 (EV-A71) is one of the common causative pathogens for hand foot and mouth disease (HFMD) affecting young children. HFMD outbreak can result in a substantial pediatric hospitalization and burden the healthcare services, especially in less-developed countries. Since the initial epidemic of predominantly EV-A71 in California in 1969, the high prevalence of HFMD in the Asia-pacific region and elsewhere around the world represents a significant morbidity in this age group. With the advent of rapid and accurate diagnostic tools, there has been a dramatic increase in the number of laboratory-confirmed EV-A71 infection over the past two decades. The population, cultural, and socioeconomic diversity among countries in the Asia-Pacific region all influence the transmission and morbidity associated with HFMD. This review summarizes the current state of epidemiology of EV-A71 in Asia-Pacific countries based on the most recent epidemiological data and available information on the prevalence and disease burden. This knowledge is important in guiding the prevention, control and future research on vaccine development of this highly contagious disease of significant socioeconomic implications in public health.


Assuntos
Surtos de Doenças , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/epidemiologia , Doença de Mão, Pé e Boca/epidemiologia , Ásia Sudeste/epidemiologia , Austrália/epidemiologia , Enterovirus Humano A/genética , Infecções por Enterovirus/virologia , Extremo Oriente/epidemiologia , Doença de Mão, Pé e Boca/virologia , Humanos , Epidemiologia Molecular
6.
Int J Infect Dis ; 87: 1-7, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31330324

RESUMO

BACKGROUND: Hand, foot and mouth disease (HFMD) is usually caused by EVA71 and CVA16 except for a few cases that are caused by non-EVA71 non-CAV16 enteroviruses. Coxsackievirus B3 (CVB3) is mostly associated with myocarditis, occasionally with HFMD. METHODS: The partial VP1 gene of enteroviruses were amplified and sequenced from 610 throat swabs from clinically confirmed HFMD children. All available CVB3 near full-length genomic and VP1 sequences were downloaded from GenBank. Phylogenetic and distance analyses were performed using MEGA 7.0. RESULTS: A total of 238 partial VP1 sequences were obtained, including 93 EVA71 (39%), 79 CAV16 (33%), 29 CVB3 (12%), 24 CVA6 (10%), and 13 other enterovirus serotypes (5.5%). CVB3 is classified into seven genotypes A-G according to phylogenetic and distance analyses. All CVB3 strains from Zhenjiang belonged to genotype A. In contrast to other genotypes that are prevalent in Europe and other regions of China, and often associated with aseptic meningitis and myocarditis, CVB3 genotype A strains identified in Zhenjiang were only detected among HFMD patients. CONCLUSIONS: This high prevalence of CVB3 genotype A among HFMD children has never been reported. This phenomenon has revealed a new epidemic trend of CVB3 among HFMD in China, and it has epidemiological implications for monitoring the epidemic risk of CVB3.


Assuntos
Enterovirus Humano A/isolamento & purificação , Doença de Mão, Pé e Boca/virologia , Sequência de Bases , Criança , Pré-Escolar , China/epidemiologia , Enterovirus/classificação , Enterovirus Humano A/classificação , Enterovirus Humano A/genética , Epidemias , Europa (Continente) , Feminino , Genótipo , Doença de Mão, Pé e Boca/epidemiologia , Humanos , Lactente , Masculino , Meningite Asséptica/epidemiologia , Meningite Asséptica/virologia , Faringe/virologia , Filogenia , Prevalência
7.
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
8.
Microb Pathog ; 134: 103568, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31195113

RESUMO

Hand, foot, and mouth disease (HFMD) is a major public health concern, especially among infants and young children. The primary pathogen of HFMD is enterovirus 71 (EV71), whose capsid assembly mechanism including capsid protein processing has been widely studied. However, some of its mechanisms remain unclear, such as the VP0 cleavage. This study aimed to identify the cleavage site of the EV71 VP0 capsid protein and to elucidate the effects of EV71 VP0 cleavage on viral infectivity and assembly. A mass spectrometry analysis indicated that the cleavage site of EV71 VP0 is located between residues Lys69 and Ser70. To analyze the importance of either residue to cleavage, we designed single mutations of Lys69, Ser70 and double mutations respectively and implemented these genomes to encapsulation. The results indicated that Ser70 is more important for VP0 cleavage and EV71 infectivity. In addition, exogenous expression of EV71 protease 2A and 3C was used to verify whether they play roles in VP0 cleavage. Analyses also showed that none of them participate in this process. This study provides novel insights into the mechanisms of EV71 capsid maturation, which may be a potential target to improve the productivity and immunogenicity of EV71 vaccines.


Assuntos
Proteínas do Capsídeo/metabolismo , Enterovirus Humano A/metabolismo , Infecções por Enterovirus/virologia , Clivagem do RNA/fisiologia , Montagem de Vírus , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Células HEK293 , Humanos , Proteínas Virais/metabolismo , Vacinas Virais
9.
Biotechnol Lett ; 41(6-7): 867-872, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31119429

RESUMO

OBJECTIVE: To construct recombinant Lactococcus lactis (L. lactis) expressing viral protein 1 (VP1) of enterovirus 71 (EV71) and evaluate its immunogenicity to be used as an oral vaccine in BALB/c mice. RESULTS: Recombinant L. lactis competent in secreting VP1 (~ 30 kDa) into the extracellular environment with the aid of the signal peptide Usp45 was produced. Enzyme-linked immunosorbent assay showed that significant VP1-specific antibody response including the production of both serum IgG and fecal IgA (p < 0.05) was elicited in BALB/c mice upon oral immunization with recombinant L. lactis. Moreover, in contrast to negative control, recombinant L. lactis induced adequate neutralizing antibodies in mouse sera (p < 0.05) as demonstrated in virus neutralization assay, whereas the presence of neutralizing antibodies in fecal samples was obvious but not significant (p > 0.05). CONCLUSIONS: Recombinant L. lactis expressing VP1 of EV71 has the potential to be used as an oral vaccine candidate. The findings may provide some preliminary evidences for further development of effective and needle-free EV71 vaccines.


Assuntos
Enterovirus Humano A/imunologia , Lactococcus lactis/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Administração Oral , Animais , Anticorpos Neutralizantes/análise , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Enterovirus Humano A/genética , Fezes/química , Imunoglobulina A/análise , Imunoglobulina G/sangue , Lactococcus lactis/genética , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
10.
Emerg Microbes Infect ; 8(1): 773-786, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31132962

RESUMO

Enterovirus 71 (EV71) is typically transmitted by the oral-faecal route and initiates infection upon crossing the intestinal mucosa. Our limited understanding of the mechanisms by which it crosses the intestinal mucosa has hampered the development of effective therapeutic options. Here, using an RNA interference screen combined with chemical inhibitors or the overexpression of dominant negative proteins, we found that EV71 entry into Caco-2 cells, a polarized human intestinal epithelial cell line, does not involve clathrin- and caveolae-dependent endocytic pathways or macropinocytosis but requires GTP-binding protein dynamin 2 and cytoskeleton remodelling. The use of siRNAs targeting endophilin family members revealed that endophlin-A2 is essential for the uptake of EV71 particles by Caco-2 cells. Subcellular analysis revealed that internalized EV71 virions largely colocalized with endophilin-A2 at cytomembrane ruffles and in the perinuclear area. Combined with viral entry kinetics, these data suggest that EV71 enters Caco-2 cells mainly via an endophilin-A2-mediated endocytic (EME) pathway. Finally, we showed that internalized EV71 virions were transported to endosomal sorting complex required for transport (ESCRT)-related multivesicular bodies (MVBs). These data provide attractive therapeutic targets to block EV71 infection.


Assuntos
Endocitose , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Internalização do Vírus , Células CACO-2 , Enterovirus Humano A/genética , Infecções por Enterovirus/virologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Mucosa Intestinal/virologia , Peptídeos e Proteínas de Sinalização Intracelular/genética
11.
BMC Infect Dis ; 19(1): 370, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046684

RESUMO

BACKGROUND: Several inactivated enterovirus-A71 (EV-A71) vaccines are currently licensed in China; however, the development of additional EV-A71 vaccines is ongoing, necessitating extensive analysis of the molecular epidemiology of the virus worldwide. Until 2012, laboratory confirmation of EV-A71 for hand, foot, and mouth disease (HFMD) and other associated diseases had not occurred in the Philippines. Because EV-A71 has been linked with cases of acute flaccid paralysis (AFP), AFP surveillance is one strategy for documenting its possible circulation in the country. To expand current knowledge on EV-A71, molecular epidemiologic analysis and genetic characterization of EV-A71 isolates were performed in this study. METHODS: A retrospective study was performed to identify and characterize nonpolio enteroviruses (NPEVs) associated with AFP in the Philippines, and nine samples were found to be EV-A71-positive. Following characterization of these EV-A71 isolates, the complete viral protein 1 (VP1) gene was targeted for phylogenetic analysis. RESULTS: Nine EV-A71 isolates detected in 2000 (n = 2), 2002 (n = 4), 2005 (n = 2), and 2010 (n = 1) were characterized using molecular methods. Genomic regions spanning the complete VP1 region were amplified and sequenced using specific primers. Phylogenetic analysis of the full-length VP1 region identified all nine EV-A71 Philippine isolates as belonging to the genogroup C lineage, specifically the C2 cluster. The result indicated a genetic linkage with several strains isolated in Japan and Taiwan, suggesting that strains in the C2 cluster identified in the Asia-Pacific region were circulating in the Philippines. CONCLUSION: The study presents the genetic analysis of EV-A71 in the Philippines. Despite some limitations, the study provides additional genetic data on the circulating EV-A71 strains in the Asia-Pacific region, in which information on EV-A71 molecular epidemiology is incomplete. Considering that EV-A71 has a significant public health impact in the region, knowledge of its circulation in each country is important, especially for formulating vaccines covering a wide variety of strains.


Assuntos
Infecções por Enterovirus/diagnóstico , Paralisia/diagnóstico , Doença Aguda , Animais , Proteínas do Capsídeo/genética , Criança , Pré-Escolar , Enterovirus Humano A/classificação , Enterovirus Humano A/genética , Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/virologia , Fezes/virologia , Febre Aftosa/diagnóstico , Febre Aftosa/virologia , Genótipo , Humanos , Lactente , Paralisia/virologia , Filipinas , Filogenia , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Estudos Retrospectivos
12.
Plasmid ; 103: 17-24, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30928703

RESUMO

In the present study, a donor plasmid derived from pUC19 and two recipient plasmids, which had been modified from the donor plasmid and contained the red fluorescence protein gene mCherry as a reporter gene downstream of the hybrid tac promoter with the -35 region deletion mutation, were constructed. The complete genome sequence of coxsackievirus A10 downstream of the T7 promoter was divided into 7 fragments and synthesized by overlap extension PCR and the DNAworks program. Using the Golden Gate cloning strategy, the 7 fragments were then cloned into the donor plasmid and transferred to the recipient plasmid upstream of the deletion mutation tac promoter in a defined order and orientation without any deletions or insertions at the junction sites. Because the -35 region of the tac promoter was introduced into the 3' end of the last fragment during construction, the hybrid promoter was reconstructed to promote expression of mCherry, which facilitated the selection of colonies with the complete genome of coxsackievirus A10 to generate an infectious cDNA clone via reverse genetic engineering.


Assuntos
Sequência de Bases , Enterovirus Humano A/genética , Genoma Viral , Plasmídeos/química , Deleção de Sequência , DNA Complementar/genética , DNA Complementar/metabolismo , Enterovirus Humano A/metabolismo , Genes Reporter , Engenharia Genética/métodos , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Genética Reversa
13.
Emerg Infect Dis ; 25(4): 788-791, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30882309

RESUMO

We investigated enterovirus A71-associated hand, foot and mouth disease in Vietnam and found that, after replacing subgenogroup C4 in 2013, B5 remained the leading cause of this disease. In contrast with previous observations, this switch did not result in an explosive outbreak, and B5 evolution was driven by negative selection.


Assuntos
Enterovirus Humano A/genética , Doença de Mão, Pé e Boca/virologia , Doença de Mão, Pé e Boca/epidemiologia , Humanos , Vietnã/epidemiologia
14.
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
15.
PLoS One ; 14(3): e0210477, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30845175

RESUMO

Enterovirus (EV) 71 is the main pathogen associated with hand-foot-mouth disease (HFMD) and can lead to the disease with severe mortality in children. Since 2009, in the Republic of Korea, an outbreak of EV71 C4a infection with neurologic involvement emerged, where in HFMD involvement was identified and central nervous system complications were reported. In this study, EV71 C4a virus-like particles (VLPs) produced by recombinant technology were generated in a baculovirus expression system. To improve the production yield, EV71 VLP was constructed using the dual promoter system baculovirus P1 and 3CD (baculo-P1-3CD), which harbored both the structural protein-encoding P1 region under the control of the polyhedron promoter and the 3CD protease gene under the regulation of the CMV-IE, lef3, gp41, or chitinase promoters to augment the level of gene transcription. Efficient VLP expression was demonstrated through optimization of incubation time and insect cell type. In addition, to evaluate the potential of VLP as a vaccine candidate, we tested the neutralizing antibodies and total anti-EV71 IgG from the purified EV71 C4a VLP serum. The recombinant EV71 VLP exhibited the morphology of self-assembled VLP, as determined by electron microscopy. Use of baculo-P1-3CD-gp41 led to a high yield (11.3mg/L < 40kDa) of VLPs in High-FiveTM cells at 3 days post-infection. Furthermore, the potential of VLP as a vaccine was evaluated through the neutralizing ability elicited by the purified EV71 VLP after immunization of BALB/c mice, which was shown to induce potent and long-lasting humoral immune responses as evidenced by the cross-neutralization titer. Our results could be used to expedite the developmental process for vaccines under clinical trials and to ensure manufacturing consistency for licensing requirements.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Enterovirus Humano A/genética , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Baculoviridae , Enterovirus Humano A/imunologia , Feminino , Engenharia Genética , Camundongos , Camundongos Endogâmicos BALB C , Células Sf9 , Vacinação , Vacinas de Partículas Semelhantes a Vírus/imunologia , Células Vero
16.
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
17.
Virol Sin ; 34(1): 66-77, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30796736

RESUMO

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, has caused periodic infection outbreaks in children in the Asia-Pacific region. In order to describe the largely unknown life cycle of EV71, the molecular basis of its virus-host interactions must first be determined. The 5' untranslated region of EV71 contains a cloverleaf-like structure and internal ribosomal entry site (IRES), which play an important role in transcription and translation of viral protein. We found that polypyrimidine tract-binding protein 1 (PTB) bound to the IRES of EV71. RNA recognition motifs 1 and 2 of PTB were responsible for its binding to the EV71 IRES. Moreover, PTB protein was shuttled from nucleus to cytoplasm after EV71 infection. Additionally, IRES activity and viral protein production were inhibited by PTB knockdown. These results suggest that PTB interacts with the EV71 IRES, and positively regulates viral protein translation.


Assuntos
Enterovirus Humano A/genética , Interações entre Hospedeiro e Microrganismos/genética , Sítios Internos de Entrada Ribossomal/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Biossíntese de Proteínas , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Ligação Proteica , RNA Viral , Proteínas Virais/metabolismo , Replicação Viral
18.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(1): 79-83, 2019 Jan 10.
Artigo em Chinês | MEDLINE | ID: mdl-30669736

RESUMO

Objective: To estimate the serotype and age-specific hospitalization burden associated with hand, foot and mouth disease (HFMD) in Anhua county of Hunan province, between October 2013 and September 2016. Methods: We collected hospitalization records of HFMD patients from 6 virological surveillance hospitals, and reimbursement records through new rural cooperative medical system from 23 township health centers to estimate the age-specific hospitalization burden of HFMD in Anhua. Combined with the results of virological surveillance, the serotype-specific hospitalization burden of HFMD in Anhua, was estimated. Results: During the three years, it was estimated that 3 541 clinical diagnosed HFMD cases, including 3 146 laboratory-confirmed HFMD cases, were hospitalized in Anhua, but only one was diaguosed as being severe. The estimated average hospitalization rate was 723/100 000(95%CI: 699/100 000-747/100 000) for clinical diagnosed HFMD and 642/100 000 (95%CI: 620/100 000-665/100 000) for laboratory-confirmed HFMD between October 2013 and September 2016. The cases caused by Cox A16 (208/100 000) and Cox A6 (202/100 000) had higher hospitalization rates compared with the cases caused by EV71 (130/100 000), Cox A10 (38/100 000) and other enterovirus (64/100 000), and the difference was statistically significant (P<0.001). HFMD-associated hospitalization rates peaked in children aged 1 year (3 845/100 000), and then decreased with age. Compared with the hospitalized HFMD caused by EV71 and Cox A16, Cox A6-associated hospitalizations mainly occurred in younger age groups (P<0.001). Conclusion: Our study revealed a substantial hospitalization burden associated with mild HFMD caused by EV71, Cox A16, Cox A6 and Cox A10, especially in young children, in Anhua.


Assuntos
Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/epidemiologia , Doença de Mão, Pé e Boca/epidemiologia , Doença de Mão, Pé e Boca/virologia , Hospitalização/estatística & dados numéricos , Criança , China/epidemiologia , Enterovirus , Enterovirus Humano A/genética , Infecções por Enterovirus/virologia , Doença de Mão, Pé e Boca/etnologia , Hospitais/estatística & dados numéricos , Humanos , Lactente , Sorogrupo
19.
BMC Immunol ; 20(1): 6, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30669993

RESUMO

BACKGROUND: Enterovirus A 71 (EV71) is a neurotropic virus that may lead to acute flaccid paralysis, encephalitis, cardiopulmonary failure or even death. No vaccine and defensive drug controlling EV71 is currently available, novel and efficient antiviral drug or vaccine is therefore urgently needed. 3Dpol (RNA-dependent RNA polymerase (RdRp)) has been an important target for anti-EV71 drug development. METHODS: A panel of monoclonal IgG antibodies (mAbs) against EV71 3Dpol were generated by traditional cell fusion methods. And the antibody affinity and specificity to EV71 3Dpol were evaluated by Enzyme-linked Immunosorbent Assay (ELISA), Indirect Fluorescent Assay (IFA) and Western blotting. Antiviral activities of these antibodies were also determined in vitro and in vivo. RESULTS: Two mAbs towards EV71 3Dpol were able to effectively suppress EV71 replication in Vero-1008 cell when intracellarly delivered. And they also dampened the RNA polymerase activity of 3Dpol in vitro. More importantly, these mAbs provided partial protection in EV71-challenged neonatal murine challenge model. CONCLUSIONS: These results showed that two of mAbs against EV71 3Dpol inhibited EV71 replication and could be utilized as promising therapeutic drug candidate.


Assuntos
Anticorpos Monoclonais/farmacologia , Antivirais/farmacologia , Enterovirus Humano A/efeitos dos fármacos , RNA Replicase/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Sítios de Ligação , Modelos Animais de Doenças , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/mortalidade , Infecções por Enterovirus/virologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Camundongos , Ligação Proteica , RNA Replicase/metabolismo
20.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30602612

RESUMO

Human enteroviruses of species A (EV-A) are the leading cause of hand-foot-and-mouth disease (HFMD). EV-A71 is frequently implicated in HFMD outbreaks and can also cause severe neurological manifestations. We investigated the molecular epidemiological processes at work and the contribution of genetic recombination to the evolutionary history of EV-A in Madagascar, focusing on the recently described EV-A71 genogroup F in particular. Twenty-three EV-A isolates, collected mostly in 2011 from healthy children living in various districts of Madagascar, were characterized by whole-genome sequencing. Eight different types were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent recent intra- and intertypic genetic exchanges between the noncapsid sequences of Madagascan EV-A isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination, with one isolate displaying a mosaic genome resulting from recent genetic exchanges with Madagascan coxsackieviruses A7 and possibly A5 and A10 or common ancestors. The engineering and characterization of recombinants generated from progenitors belonging to different EV-A types or EV-A71 genogroups with distantly related nonstructural sequences indicated a high level of permissiveness for intertypic genetic exchange in EV-A. This permissiveness suggests that the primary viral functions associated with the nonstructural sequences have been highly conserved through the diversification and evolution of the EV-A species. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify surveillance of EV-A circulation and HFMD cases to prevent possible outbreaks due to emerging strains.IMPORTANCE Human enteroviruses of species A (EV-A), including EV-A71, are the leading cause of hand-foot-and-mouth disease (HFMD) and may also cause severe neurological manifestations. We investigated the circulation and molecular evolution of EV-A in Madagascar, focusing particularly on the recently described EV-A71 genogroup F. Eight different types, collected mostly in 2011, were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent genetic exchanges between the different types of isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination. The engineering and characterization of recombinants involving progenitors belonging to different EV-A types indicated a high degree of permissiveness for genetic exchange in EV-A. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify the surveillance of EV-A circulation to prevent possible HFMD outbreaks due to emerging strains.


Assuntos
Enterovirus Humano A/genética , Recombinação Genética/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Pré-Escolar , Surtos de Doenças , Infecções por Enterovirus/virologia , Evolução Molecular , Genoma Viral/genética , Genótipo , Células HEK293 , Doença de Mão, Pé e Boca/genética , Doença de Mão, Pé e Boca/virologia , Humanos , Madagáscar , Epidemiologia Molecular , Permissividade , Filogenia , Células Vero , Sequenciamento Completo do Genoma/métodos
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