RESUMO
INTRODUCTION: Virus-like particles (VLPs), self-assembled multiprotein structures, can stimulate robust immune responses due to their structural similarity to native virions that allow the presentation of multiple copies of the target epitopes. Utilizing VLPs as vaccine platforms to present exogenous antigens is a promising and challenging approach in the vaccine development field. This study investigates the potential of the truncated hepatitis E virus (HEV) capsid as a VLP platform to present foreign antigens. METHODS: The S and M domains of the HEV capsid protein were selected as the optimal carrier (CaSM). The exogenous antigen Seq8 containing 3 neutralizing epitopes from 3 different foot-and-mouth disease virus (FMDV) strains was linked to the C-terminal of CaSM to construct a chimeric VLP (CaSM-Seq8). The chimeric particles were produced in Escherichia coli, and their morphology, physicochemical properties, antigenicity, and immunogenicity were analyzed. RESULTS: Morphological analysis showed that CaSM-Seq8 self-assembled into VLPs similar to CaSM VLPs (â¼26 nm in diameter) but smaller than native HEV virions. Further, the thermal stability and the resistance to enzymatic proteolysis of Seq8 were enhanced when it was attached to the CaSM carrier. The antigenicity analysis revealed a more robust reactivity against anti-FMDV antibodies when Seq8 was presented on CaSM particles. Upon injection into mice, FMDV-specific IgGs induced by CaSM-Seq8 appeared earlier, increased faster, and maintained higher levels for a longer time than those induced by Seq8 alone or the inactivated FMDV vaccine. CONCLUSION: This study demonstrated the potential of utilizing the truncated HEV capsid as an antigen-presenting platform for the development of chimeric VLP immunogens.
Assuntos
Vírus da Febre Aftosa , Vírus da Hepatite E , Vacinas de Partículas Semelhantes a Vírus , Animais , Capsídeo , Proteínas do Capsídeo/genética , Vírus da Hepatite E/genética , Camundongos , Desenvolvimento de Vacinas , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
BACKGROUND: Zoonotic hepatitis E virus (HEV) infection emerged as a serious threat in the industrialized countries. The aim of this study is exploring a new approach for the control of zoonotic HEV in its main host (swine) through the design and development of an economically interesting chimeric vaccine against HEV and against a devastating swine infection: the foot-and-mouth disease virus (FMDV) infection. RESULTS: First, we adopted a computational approach for rational and effective screening of the different HEV-FMDV chimeric proteins. Next, we further expressed and purified the selected chimeric immunogens in Escherichia coli (E. coli) using molecular cloning techniques. Finally, we assessed the antigenicity and immunogenicity profiles of the chimeric vaccine candidates. Following this methodology, we designed and successfully produced an HEV-FMDV chimeric vaccine candidate (Seq 8-P222) that was highly over-expressed in E. coli as a soluble protein and could self-assemble into virus-like particles. Moreover, the vaccine candidate was thermo-stable and exhibited optimal antigenicity and immunogenicity properties. CONCLUSION: This study provides new insights into the vaccine development technology by using bioinformatics for the selection of the best candidates from larger sets prior to experimentation. It also presents the first HEV-FMDV chimeric protein produced in E. coli as a promising chimeric vaccine candidate that could participate in reducing the transmission of zoonotic HEV to humans while preventing the highly contagious foot-and-mouth disease in swine.
Assuntos
Febre Aftosa/prevenção & controle , Hepatite E , Proteínas Recombinantes de Fusão , Vacinas Virais , Animais , Hepatite E/prevenção & controle , Hepatite E/veterinária , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Suínos , Vacinas Virais/biossíntese , Vacinas Virais/imunologiaRESUMO
BACKGROUND: The hepatitis E virus (HEV) is the causative pathogen of hepatitis E, a global public health concern. HEV comprises 8 genotypes with a wide host range and geographic distribution. This study aims to determine the genetic factors influencing the molecular adaptive changes of HEV open reading frames (ORFs) and estimate the HEV origin and evolutionary history. RESULTS: Sequences of HEV strains isolated between 1982 and 2017 were retrieved and multiple analyses were performed to determine overall codon usage patterns, effects of natural selection and/or mutation pressure and host influence on the evolution of HEV ORFs. Besides, Bayesian Coalescent Markov Chain Monte Carlo (MCMC) Analysis was performed to estimate the spatial-temporal evolution of HEV. The results indicated an A/C nucleotide bias and ORF-dependent codon usage bias affected mainly by natural selection. The adaptation of HEV ORFs to their hosts was also ORF-dependent, with ORF1 and ORF2 sharing an almost similar adaptation profile to the different hosts. The discriminant analysis based on the adaptation index suggested that ORF1 and ORF3 could play a pivotal role in viral host tropism. CONCLUSION: In this study, we estimate that the common ancestor of the modern HEV strains emerged ~ 6000 years ago, in the period following the domestication of pigs. Then, natural selection played the major role in the evolution of the codon usage of HEV ORFs. The significant adaptation of ORF1 of genotype 1 to humans, makes ORF1 an evolutionary indicator of HEV host speciation, and could explain the epidemic character of genotype 1 strains in humans.
Assuntos
Evolução Molecular , Vírus da Hepatite E/genética , Códon , Vírus da Hepatite E/classificação , Mutação , Nucleotídeos/análise , Fases de Leitura Aberta , Filogenia , Seleção Genética , Proteínas Virais/genéticaRESUMO
INTRODUCTION AND OBJECTIVES: This study was undertaken to demonstrate a promising approach for detection and differentiation the serum immunoglobulin G (IgG) against hepatitis E virus (anti-HEV IgG) using a competitive binding assay established with known genotype-specific monoclonal antibodies (mAbs) 2B1 and 4C5. MATERIALS AND METHODS: The mAb 2B1 derived from genotype 1 hepatitis E virus (HEV) antigen and specifically reacted with genotype 1, 2 antigens; 4C5 induced by genotype 4 HEV antigen was specific to genotypes 3, 4 antigens. The 2B1 and 4C5 were labeled with Horseradish peroxidase (HRP), respectively. Subsequently, the titers of coated antigens and HRP-conjugated mAbs for establishment of competitive binding assay were determined by enzyme linked immunosorbent assay (ELISA). And then, the competitive binding assay was performed to assess the inhibition percentage of mAbs binding to antigens inhibited by different genotypes anti-HEV IgG. RESULTS: The results of competitive binding assay revealed that genotype 1 anti-HEV IgG could inhibit the binding of mAb 2B1 to genotype 1 antigen more strongly than that of mAb 4C5 to genotype 4 antigen. Whereas, the genotype 3 or 4 anti-HEV IgG could inhibit the binding of mAb 4C5 to genotype 4 antigen more remarkably than that of mAb 2B1 to genotype 1 antigen. CONCLUSIONS: These findings provided us a valuable approach for detection and differentiation the HEV infection derived from genotypes 1, 2 (human) or genotypes 3, 4 (zoonosis).
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Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Anti-Hepatite/imunologia , Vírus da Hepatite E/imunologia , Hepatite E/diagnóstico , Imunoglobulina G/imunologia , Anticorpos Monoclonais , Genótipo , Vírus da Hepatite E/genética , Peroxidase do Rábano Silvestre , HumanosRESUMO
Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA.
Assuntos
Vírus da Hepatite E/genética , Vírus da Hepatite E/isolamento & purificação , Hepatite E/diagnóstico , RNA Viral/sangue , Reação em Cadeia da Polimerase em Tempo Real/métodos , Hepatite E/virologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Sensibilidade e Especificidade , Carga Viral , Organização Mundial da SaúdeRESUMO
BACKGROUND: Viral protein expression in Escherichia coli (E. coli) is a powerful tool for structural/functional studies as well as for vaccine and diagnostics development. However, numerous factors such as codon bias, mRNA secondary structure and nucleotides distribution, have been indentified to hamper this heterologous expression. RESULTS: In this study, we combined computational and biochemical methods to analyze the influence of these factors on the expression of different segments of hepatitis E virus (HEV) ORF 2 protein and hepatitis B virus surface antigen (HBsAg). Three out of five HEV antigens were expressed while all three HBsAg fragments were not. The computational analysis revealed a significant difference in nucleotide distribution between expressed and non-expressed genes; and all these non-expressing constructs shared similar stable 5'-end mRNA secondary structures that affected the accessibility of both Shine-Dalgarno (SD) sequence and start codon AUG. By modifying the 5'-end of HEV and HBV non-expressed genes, there was a significant increase in the total free energy of the mRNA secondary structures that permitted the exposure of the SD sequence and the start codon, which in turn, led to the successful expression of these genes in E. coli. CONCLUSIONS: This study demonstrates that the mRNA secondary structure near the start codon is the key limiting factor for an efficient expression of HEV ORF2 proteins in E. coli. It describes also a simple and effective strategy for the production of viral proteins of different lengths for immunogenicity/antigenicity comparative studies during vaccine and diagnostics development.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , RNA Mensageiro/química , Proteínas Virais/genética , Códon , Códon de Iniciação , Conformação de Ácido Nucleico , Proteínas Recombinantes/genéticaRESUMO
AIMS: To confirm the different characteristics of genotype-specific and common neutralizing epitopes of hepatitis E virus (HEV). METHODS: A competitive binding assay was established with known genotype-common neutralizing monoclonal antibodies (mAbs) 3G1 and 5G5 as well as genotype-specific neutralizing mAbs 2B1 and 4C5. HEV ORF2 recombinant p166W01 derived from genotype 1 and p166Chn derived from genotype 4 were used as coated antigens, to determine whether the mAbs recognize independent, similar, or overlapping epitopes. mAbs were produced, purified, and conjugated with horseradish peroxidase (HRP). HRP-conjugated 2B1 could react only with p166W01 but not p166Chn, HRP-conjugated 4C5 could react only with p166Chn but not p166W01, while HRP-conjugated 3G1 and 5G5 could react both with p166W01 and p166Chn. Thus, competitive binding assays were performed successively using p166W01 and p166Chn antigen. RESULTS AND CONCLUSION: The results of competitive binding assays revealed that the binding of HRP-conjugated 2B1 to p166W01 could not be inhibited by 5G5 or 3G1. Similarly, the binding of HRP-conjugated 4C5 to p166Chn could not be inhibited by 5G5 or 3G1. However, the mAbs 5G5 and 3G1 blocked each other's binding to p166W01 and p166Chn, suggesting that common and genotype-specific neutralizing mAbs recognize independent epitopes.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Antígenos Virais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Vírus da Hepatite E/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/genética , Ligação Competitiva , Mapeamento de Epitopos/métodos , Epitopos/genética , Vírus da Hepatite E/genética , Peroxidase do Rábano Silvestre/química , Humanos , Hibridomas/química , Hibridomas/imunologia , Imunoconjugados/química , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Angong Niuhuang Wan (AGNHW, ), is a classical medicinal formula in Traditional Chinese Medicine (TCM) that has been appreciated for its neuroprotective properties in ischemic cerebral injuries, yet its intricate mechanisms remain only partially elucidated. AIMS: This study leverages advanced Mass cytometry (CyTOF) to analyze AGNHW's multifaceted immunomodulation effects in-depth, emphasizing previously underexplored areas. RESULTS: AGNHW mitigated monocyte-derived macrophages (MoDM) infiltration in the brain, distinguishing its effects on those from microglia. While the vehicle group exhibited elevated inflammatory markers like CD4, CD8a, and CD44 in ischemic brains, the AGNHW-treated group attenuated their expressions, indicating AGNHW's potential to temper the post-ischemic inflammatory response. Systemically, AGNHW modulated fundamental immune cell dynamics, notably augmenting CD8+ T cells, B cells, monocytes, and neutrophil counts in the peripheral blood under post-stroke conditions. Intracellularly, AGNHW exhibited its targeted modulation of the signaling pathways, revealing a remarked inhibition of key markers like IκBα, indicating potential suppression of inflammatory responses in ischemic brain injuries. CONCLUSION: This study offers a comprehensive portrait of AGNHW's immunomodulation effects on ischemic stroke, illuminating its dual sites of action-both cerebral and systemic-and its nuanced modulation of cellular and molecular dynamics.
Assuntos
Medicamentos de Ervas Chinesas , AVC Isquêmico , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , AVC Isquêmico/imunologia , AVC Isquêmico/tratamento farmacológico , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Citometria de Fluxo/métodos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismoRESUMO
Epileptogenesis, arising from alterations in synaptic strength, shares mechanistic and phenotypic parallels with memory formation. However, direct evidence supporting the existence of seizure memory remains scarce. Leveraging a conditioned seizure memory (CSM) paradigm, we found that CSM enabled the environmental cue to trigger seizure repetitively, and activating cue-responding engram cells could generate CSM artificially. Moreover, cue exposure initiated an analogous process of memory reconsolidation driven by mammalian target of rapamycin-brain-derived neurotrophic factor signaling. Pharmacological targeting of the mammalian target of rapamycin pathway within a limited time window reduced seizures in animals and interictal epileptiform discharges in patients with refractory seizures. Our findings reveal a causal link between seizure memory engrams and seizures, which leads us to a deeper understanding of epileptogenesis and points to a promising direction for epilepsy treatment.
Assuntos
Eletroencefalografia , Epilepsia , Animais , Humanos , Convulsões/etiologia , Sirolimo , Serina-Treonina Quinases TOR , MamíferosRESUMO
During 2001-2011, hepatitis E virus (HEV) was found in the blood of patients in Nanjing, China. All HEV-positive patients had virus genotype 4; subgenotype 4a was predominant. The effective population of HEV in Nanjing increased in ≈1980 and continued until ≈2003 when it plateaued.
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Genótipo , Vírus da Hepatite E/genética , Hepatite E/epidemiologia , China/epidemiologia , Evolução Molecular , Vírus da Hepatite E/classificação , Humanos , Dados de Sequência Molecular , Tipagem Molecular , Estações do Ano , SorotipagemRESUMO
UNLABELLED: The interferon (IFN) system is integral to the host response against viruses, and many viruses have developed strategies to overcome its antiviral effects. The effects of hepatitis E virus (HEV), the causative agent of hepatitis E, on IFN signaling have not been investigated primarily because of the nonavailability of an efficient in vitro culture system or small animal models of infection. We report here the generation of A549 cell lines persistently infected with genotype 3 HEV, designated as HEV-A549 cells and the effects HEV has on IFN-α-mediated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. Treatment of HEV-A549 cells with 250, 500, and 1000 U/mL of IFN-α for 72 hours showed a dose-dependent reduction in HEV RNA levels by 10%, 20%, and 50%, respectively. IFN-α-stimulated genes coding for the antiviral proteins dsRNA-activated protein kinase (PKR) and 2',5'-oligoadenylate synthetase (2',5'-OAS) were down-regulated in IFN-α-treated HEV-A549 cells. HEV infection also prevented IFN-α-induced phosphorylation of STAT1. Regulation of STAT1 by HEV was specific, as phosphorylation of STAT2, tyrosine kinase (Tyk) 2, and Jak1 by IFN-α was unaltered. Additionally, STAT1 levels were markedly increased in HEV-A549 cells compared with naive A549 cells. Furthermore, binding of HEV open reading frame (ORF)3 protein to STAT1 in HEV-A549 cells was observed. HEV ORF3 protein alone inhibited IFN-α-induced phosphorylation of STAT1 and down-regulated the IFN-α-stimulated genes encoding PKR, 2',5'-OAS, and myxovirus resistance A. CONCLUSION: HEV inhibits IFN-α signaling through the regulation of STAT1 phosphorylation in A549 cells. These findings have implications for the development of new strategies against hepatitis E.
Assuntos
Vírus da Hepatite E/genética , Interferon-alfa/farmacologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/fisiologia , Células Cultivadas/efeitos dos fármacos , Regulação para Baixo , Imunofluorescência , Regulação Viral da Expressão Gênica , Hepatite E/genética , Hepatite E/fisiopatologia , Vírus da Hepatite E/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Immunoblotting , Interferon-alfa/metabolismo , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Fosforilação , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fator de Transcrição STAT1/genética , Sensibilidade e Especificidade , Transdução de Sinais/genética , Transfecção , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Rabbit hepatitis E virus (HEV) in China may represent a novel HEV genotype, although no consensus has been reached. It is unclear whether the ORF2 capsid protein containing the immunodominant epitopes from rabbit HEV differs from those of human HEV. In this study, 661 bile samples collected from domestic rabbits in Jiangsu province, eastern China were amplified by RT-nPCR using a set of HEV universal ORF2 primers. All 42 (6.4%) positive PCR products were sequenced. Phylogenetic analysis using the ORF2 sequences of 557 bp in length showed the Jiangsu isolates were separate from HEV genotypes 1, 2, 3, 4, avian HEV and rat HEV, and clustered together with rabbit HEV sequences. These 42 isolates were divided into five branches including two newly identified in the present study. Comparison with rabbit HEV sequences from China available in GenBank, using a 298 bp ORF2 segment, showed these sequences clustered together into a unique rabbit HEV clade, and were divided into eight sub-branches with high genetic heterogeneity. In addition, 267 serum samples collected from domestic rabbits, serial serum samples from two rhesus monkeys experimentally infected with HEV genotype 1 or 4, and serial serum samples from two New-Zealand rabbits infected experimentally with rabbit HEV were tested simultaneously by EIA using recombinant truncated ORF2 capsid proteins derived from rabbit and human HEV. The virtually identical results obtained suggest that rabbit and human HEV ORF2 antigens contain very similar immunodominant epitopes. All these data are helpful to identify the biological characteristics of the newly identified rabbit HEV.
Assuntos
Vírus da Hepatite E/classificação , Vírus da Hepatite E/isolamento & purificação , Hepatite E/veterinária , Doenças dos Roedores/virologia , Animais , China , Análise por Conglomerados , Epitopos/imunologia , Genótipo , Anticorpos Anti-Hepatite/sangue , Hepatite E/virologia , Vírus da Hepatite E/genética , Vírus da Hepatite E/imunologia , Humanos , Macaca mulatta , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Sorotipagem , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Objective: This study aimed to evaluate whether CT angiography (CTA) manifestations in anterior cerebral artery a1 segment (A1) were related to the hemodynamics in patients with internal carotid artery stenosis (ICAS). Methods: A total of 97 cases were selected. The degree of ICAS and symmetry of A1 were evaluated by CTA examination. Hemodynamic indexes were detected by transcranial Doppler (TCD). The differences in CTA presentations of A1 and hemodynamics between the vessels on the stenotic and contralateral sides were analyzed according to the different degrees of stenosis. The degree of ICAS according to the different manifestations of A1 and the hemodynamics of A1's adjacent vessels were also analyzed. Results: In the case of unilateral ICAS, the difference in Vm of A1 between the stenotic and the contralateral side was the most significant relative to the stenosis degree. When unilateral ICAS was ≥70%, the presentation of A1 on the stenotic side was more slender or non-visualized compared to that on the contralateral side, while in cases with unilateral stenosis <70% or bilateral stenosis with a similar degree of stenosis, A1 were mainly symmetrical. When A1 on the side of ICAS was slender or non-visualized, the Vm of A1 was significantly slower than that on the contralateral side (P < 0.001). Conclusion: The CTA manifestations of A1 on the side of ICAS embodied the overall changes of the intracranial hemodynamics after ICAS. A combination of TCD and CTA examination of A1 can assist in judging the location and degree of ICAS.
RESUMO
Ganoderic acid T (GA-T) is an important triterpene of Ganoderma lucidum, which is utilized to treat viral infections. Sendai virus (SeV) is widely studied to determine the molecular biological characteristics of RNA viruses and employed to elucidate the mechanisms governing the innate immune response. However, the comprehensive mechanism governing the antiviral effects of GA-T against SeV infection remains unknown. In this study, SeV-infected host cells were treated with 16.3 µM GA-T, subsequently RNA-seq analysis was performed to screen the differentially expressed genes (DEGs). The RNA-seq data showed that GA-T treatment upregulated 934 DEGs and downregulated 1283 DEGs against viral infection, in particularly, IFNGR1, IL1A, and IL1R1 were upregulated, and mTOR, SMAD3, IFNL2 and IFNL3 were decreased. GO and KEGG analysis illustrated that DEGs were clustered in mTOR and IL-17 signalling pathways. Protein-protein interaction network analysis indicated the high degree of nodes, such as CXCL8, CSF2, CXCL1 and MYD88. Our results indicated that GA-T exerted its antiviral pharmacological effects through inhibition of the mTOR signalling pathway and adjustment of innate immunity system and the inflammatory response involving the IL-17 signalling pathway. Our results may help to elucidate the potential functions and underlying mechanisms governing the antiviral effects of GA-T.
Assuntos
Perfilação da Expressão Gênica , Interleucina-17 , Serina-Treonina Quinases TOR , Antivirais , Biologia Computacional , TranscriptomaRESUMO
Hand, foot, and mouth disease (HFMD) is caused mainly by enterovirus 71 (EV71) and other enteroviruses (EVs) such as Coxsackie A16 in China. EV71 infection can lead to severe clinical manifestations and even death. Other EVs, however, generally cause mild symptoms. Thus, early and accurate distinction of EV71 from other EVs for HFMD will offer significant benefits. A one-step, single tube, duplex RT-PCR assay is described in the present study to detect simultaneously EV71 and other EVs. The primers used for the duplex RT-PCR underwent screening and optimization. The detection threshold was 0.001 TCID(50)/ml for EV71 and 0.01 TCID(50)/ml for other EVs. The positive rate of enterovirus detection in 165 clinical samples reached 68.5%, including 46.1% for EV71 and 22.4% for other EVs. Of all the severe HFMD cases, EV71 was responsible for 85.3% cases. The positive rate of EV71 fell markedly by day 8 after onset. In addition, sequencing of EV71 specific amplicons from duplex RT-PCR revealed that C4a was the predominant subgenotype of EV71 circulating in Nanjing, China. The accuracy and reliability of the assay suggest strongly that the one-step, single tube, duplex RT-PCR will be useful for early diagnosis and monitoring of EV71 and other EV infections.
Assuntos
Enterovirus Humano A/isolamento & purificação , Doença de Mão, Pé e Boca/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virologia/métodos , Animais , China , Primers do DNA/genética , Enterovirus Humano A/classificação , Enterovirus Humano A/genética , Genótipo , Humanos , Dados de Sequência Molecular , Prevalência , RNA Viral/genética , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Zoonotic hepatitis E virus (HEV) infection is an emerging global public health concern. It is usually transmitted to humans from domestic pigs (main host). Since virus-like particles (VLPs) exhibit unique structural and immunological characteristics that make them of momentous applications in vaccine development, the purpose of the present study was the production of immunogenic chimeric VLPs as vaccine candidates for the control of zoonotic HEV in its main host and the prevention of porcine circovirus associated disease, a multi-factorial disease with major economic repercussions on global pig industry. An immuno-informatics approach was applied for the design and screening of new chimeric antigens presenting the dominant immunogenic domains of both HEV and porcine circovirus 2 (PCV2). Then, using molecular cloning techniques, the chimeric proteins were expressed in Escherichia coli. After purification, full characterization of the physicochemical, morphological, and immunological properties of the target proteins has been conducted. The chimeric immunogens were successfully overexpressed and after the optimization of the expression conditions, 5 chimeric proteins were efficiently purified under native conditions. The purified HEV-PCV2 chimeric proteins were found thermo-stable and able to self-assemble into spherical virus-like particles. Four HEV-PCV2 chimeric proteins have displayed optimal antigenicity and immunogenicity properties, with the nPCV2cp-p166 chimeric immunogen slightly outranking the other designed proteins. In conclusion, this study reports the production of stable HEV-PCV2 chimeric VLPs that exhibited optimal antigenicity and immunogenicity and thus with potential applications in diagnostics and vaccine development. Besides, this study provides a reproducible approach for the design, assessment, and production of chimeric antigens.
Assuntos
Infecções por Circoviridae , Circovirus , Vírus da Hepatite E , Hepatite E , Doenças dos Suínos , Vacinas Virais , Animais , Proteínas do Capsídeo , Infecções por Circoviridae/veterinária , Circovirus/genética , Hepatite E/prevenção & controle , Hepatite E/veterinária , Vírus da Hepatite E/genética , Proteínas Recombinantes de Fusão/genética , Suínos , Doenças dos Suínos/prevenção & controleRESUMO
Hepatitis E is recognized as a zoonosis, and swine are known reservoirs, but how broadly enzootic its causative agent, hepatitis E virus (HEV), is remains controversial. To determine the prevalence of HEV infection in animals, a serological assay with capability to detect anti-HEV-antibody across a wide variety of animal species was devised. Recombinant antigens comprising truncated capsid proteins generated from HEV-subgenomic constructs that represent all four viral genotypes were used to capture anti-HEV in the test sample and as an analyte reporter. To facilitate development and validation of the assay, serum samples were assembled from blood donors (n = 372), acute hepatitis E patients (n = 94), five laboratory animals (rhesus monkey, pig, New Zealand rabbit, Wistar rat, and BALB/c mouse) immunized with HEV antigens, and four pigs experimentally infected with HEV. The assay was then applied to 4,936 sera collected from 35 genera of animals that were wild, feral, domesticated, or otherwise held captive in the United States. Test positivity was determined in 457 samples (9.3%). These originated from: bison (3/65, 4.6%), cattle (174/1,156, 15%), dogs (2/212, 0.9%), Norway rats (2/318, 0.6%), farmed swine (267/648, 41.2%), and feral swine (9/306, 2.9%). Only the porcine samples yielded the highest reactivities. HEV RNA was amplified from one farmed pig and two feral pigs and characterized by nucleotide sequencing to belong to genotype 3. HEV infected farmed swine primarily, and the role of other animals as reservoirs of its zoonotic spread appears to be limited.
Assuntos
Doenças Endêmicas , Vírus da Hepatite E/isolamento & purificação , Hepatite E/veterinária , Animais , Antígenos Virais , Genótipo , Anticorpos Anti-Hepatite/sangue , Hepatite E/epidemiologia , Vírus da Hepatite E/classificação , Vírus da Hepatite E/genética , Humanos , Dados de Sequência Molecular , RNA Viral/genética , Análise de Sequência de DNA , Estudos Soroepidemiológicos , Estados Unidos/epidemiologiaRESUMO
E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.
Assuntos
Células da Medula Óssea/citologia , Caderinas/genética , Proliferação de Células , Leucemia/patologia , Células da Medula Óssea/metabolismo , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Leucemia/metabolismo , RNA Interferente Pequeno/metabolismo , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Objective: To study whether there is a difference in peak and mean blood flow velocity between the left and right major cerebral vessels in patients with epilepsy. Methods: Sixteen patients with epilepsy underwent FDG18-PET-CT (PET) scan and electroencephalogram (EEG) examinations. Transcranial Doppler (TCD) was used to detect the peak flow velocity (PFV), mean flow velocity (MFV), and other hemodynamic indicators of bilateral anterior, middle, and posterior cerebral arteries in each patient. According to different patterns of the PET or interictal EEG, the differences in PFV, and MFV of corresponding vessels on both sides under different patterns were compared. Results: According to the PET of the low-metabolism region corresponding to the supplying artery, the PFV and MFV of the supplying artery in the low-metabolism region were lower than the value of the corresponding contralateral vessel. The PFV and MFV on the low metabolic side of PET were lower than that of the corresponding vessels on the opposite side. The PFV and MFV on the discharge side of interictal EEG were also lower than the PFV and MFV of the corresponding vessels on the opposite side. The MFV of posterior cerebral artery on the low metabolic side of PET or the interictal discharge side was significantly different from that of the contralateral vessels (P < 0.05). However, the other aforementioned differences in PFV and MFV did not achieve statistical significance. Conclusion: In epileptic patients, the PFV and MFV of main cerebral vessels on the PET hypometabolized side or the interictal discharge side was lower than that of corresponding vessels on the contralateral side. To some extent, the difference in the MFV of PCA between the bilateral sides can facilitate the lateral diagnosis of the epileptogenic zone.
RESUMO
In a continuous effort to develop effective vaccines against hepatitis E (HE), oral vaccine nanoparticles using the truncated capsid protein p146 (aa460-605) are formulated and characterized. To improve the immunogenicity of p146, chitosan nanoparticles (CSNPs) are used as a mucosal delivery system. Next, the physical-chemical properties, cytotoxic effects in vitro, and immunogenicity in mice of the produced NPs are analyzed. The results show that the produced CS/p146 NPs are stable and well dispersive and display a near-spherical shape with a mean size of 200-300 nm. The findings also demonstrate high encapsulation efficiency (65-73.9%) and loading capacity (27.7-67.5%) of the formulated nanoparticles. Further, the CS/p146 NPs exhibit low cytotoxicity and an obvious sustained-release effect in vitro. Immunogenicity experiments in mice indicate that CS/p146 NPs can induce antigen-specific systemic and mucosal immune responses higher than the purified p146 do. Besides, the expression levels and mRNA transcription of Interleukin (IL)-4 in spleen cells of CS/p146 NPs-immunized mice are higher than those of p146, indicating that a Th2-mediated cellular immune response is activated by the CS/p146 NPs. Overall, the synthesized CS/p146 NPs display promising properties as a potential HE oral vaccine candidate.