RESUMEN

Background: To determine the non-inferiority of the seven common serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) in the 13-valent pneumococcal conjugate vaccine (PCV13) with each serotype conjugated to a tetanus toxoid carrier protein and adsorbed on aluminum phosphate and the superiority of its six additional serotypes (1, 3, 5, 6A, 7F, and 19A) to the serotypes in the PCV7. Methods: Participants were evenly randomized in a 1:1 ratio into either the PCV13 or PCV7 groups, to receive three doses of the vaccine at the age of 3, 4, and 5 months, respectively, and a booster dose between 12 and 15 months of age. Serotype-specific antibodies were measured using a standardized enzyme-linked immunosorbent assay (ELISA) and opsonophagocytic activity (OPA) microcolony assay method. Results: A total of 1,040 healthy infants were enrolled. All the seven common serotypes in the PCV13 were non-inferior to those in the PCV7 in terms of the serotype-specific IgG production induced; however, non-inferiority was not shown for serotype 6B after the infant series. The proportion of subjects who reached OPA antibody titers ≥ 1:8 in the PCV13 group was 89.25% or higher. Local reactions and systemic events were mild or moderate in severity and similar between the two groups. No new safety signals were observed. Conclusion: The newly developed PCV13 was immunogenic for all serotypes and had a comparable safety profile to the marketed PCV7.

RESUMEN

Circular RNA (circRNAs) may mediate mRNA expression as miRNA sponge. Since the community has paid more attention on circRNAs, a lot of circRNA databases have been developed for plant. However, a comprehensive collection of circRNAs in crop response to abiotic stress is still lacking. In this work, we applied a big-data approach to take full advantage of large-scale sequencing data, and developed a rich circRNA resource: CropCircDB for maize and rice, later extending to incorporate more crop species. We also designed a metric: stress detections score, which is specifically for detecting circRNAs under stress condition. In summary, we systematically investigated 244 and 288 RNA-Seq samples for maize and rice, respectively, and found 38 785 circRNAs in maize, and 63 048 circRNAs in rice. This resource not only supports user-friendly JBrowser to visualize genome easily, but also provides elegant view of circRNA structures and dynamic profiles of circRNA expression in all samples. Together, this database will host all predicted and validated crop circRNAs response to abiotic stress.

Asunto(s)

Productos Agrícolas , Bases de Datos de Ácidos Nucleicos , ARN Circular , ARN de Planta , Estrés Fisiológico , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo

RESUMEN

OBJECTIVE: To construct and characterize EGFP reporter gene labeled Sindbis virus (SINV). METHODS: The reporter gene EGFP was inserted into the genome of infectious clone pBR-XJ160 by using multi-fusion long fragment PCR method. Then apply reverse genetic manipulation technique to rescue and obtain EGFP labeled SINV. RESULTS: We successively obtained labeled SINV, which has good fluorescent expression characteristics and genetic stability. CONCLUSION: The labeled virus can be seen in living cells and living body, and this serves as a good tool for cell and tissue tropism and biological function study of viruses. This study laid a foundation for further studying the cell tropism, biological functions and infection mechanism of SINV.

Asunto(s)

Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Virus Sindbis/genética , Secuencia de Bases , Datos de Secuencia Molecular

RESUMEN

Based on an infectious clone of Sindbis-like virus XJ-160, recombinant vectors containing a reporter gene (enhanced green fluorescence protein [EGFP] or Gaussia luciferase [GLUC]) were constructed by placing the reporter gene cassette containing the subgenomic promoter behind the 3' terminus of the viral structural protein gene. EGFP/GLUC-tagged Sindbis-like viruses were rescued in BHK-21 cells transfected with transcripts produced from the recombinant vectors. EGFP expression and strong luciferase activity were detected in BHK-21 cells infected with repeated passages of the EGFP/GLUC-tagged viruses, revealing the genetic stability of the chimeric viruses. The EGFP/GLUC-tagged Sindbis viruses reported will contribute to the assessment of viral replication and proliferation, tracking and elucidating Alphavirus-host interactions, and screening for antiviral compounds.

Asunto(s)

Genes Reporteros , Virus Sindbis/genética , Infecciones por Alphavirus , Animales , Línea Celular , Cricetinae , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Luciferasas/genética , Regiones Promotoras Genéticas , Virus Sindbis/fisiología , Replicación Viral

RESUMEN

OBJECTIVE: To construct the recombinant virus-like particles containing HCV envelope glycoprotein E1E2 based on sindbis virus vector. METHODS: The gene encoding HCV envelope glycoprotein E1E2 was cloned into sindbis virus vector to construct recombinant plasmids pBR-XJE1E2 and pVA-XJE1E2, and transfect them into BHK-21 cells to obtain recombinant virus-like particles. The expression of E1 and E2 protein were verified by Western Blot and indirect immunofluorescent assay (IFA). RESULTS: The results of restriction enzyme digestion, PCR and sequencing analysis showed that the recombinant plasmids were constructed successfully. And the results of RT-PCR, Western blotting and IFA detection showed that the transfect cells could package HCV-like particles of expressing structural proteins E1E2. CONCLUSION: The recombinant expression plasmids pBR-XJE1E2 and pVA-XJE1E2 based on sindbis virus vector could package HCV-like particles in eukaryotic cell, which provides a foundation for further study of its in vivo animal immune response.

Asunto(s)

Hepacivirus/genética , Virus Sindbis/genética , Proteínas del Envoltorio Viral/genética , Animales , Células Cultivadas , Cricetinae , Vectores Genéticos , Plásmidos , Recombinación Genética

RESUMEN

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.

Asunto(s)

Infecciones por Alphavirus/diagnóstico , Alphavirus/aislamiento & purificación , Genes Reporteros/genética , Ensayo de Placa Viral/métodos , Alphavirus/crecimiento & desarrollo , Infecciones por Alphavirus/genética , Ingeniería Genética/métodos , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Límite de Detección , Sensibilidad y Especificidad , Virus Sindbis/genética , Proteínas no Estructurales Virales/genética

RESUMEN

To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV) promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP) or Gaussia luciferase (G.luc) were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

Asunto(s)

Ingeniería Genética/métodos , Vectores Genéticos , Glicoproteínas/metabolismo , Virus Sindbis/crecimiento & desarrollo , Virus Sindbis/genética , Proteínas Virales/metabolismo , Genes Reporteros , Glicoproteínas/genética , Humanos , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virales/genética

RESUMEN

Heparan sulfate (HS) is ubiquitously expressed on the surfaces and in the extracellular matrix of virtually all cell types, making it an ideal receptor for viral infection. Compared with wild-type viruses, cell culture-adapted laboratory strains exhibit more efficient binding to cellular HS receptors. HS-binding viruses are typically cleared faster from the circulation and cause lower viremia than their non-HS-binding counterparts, suggesting that the HS-binding phenotype is a tissue culture adaptation that lowers virus fitness in vivo. However, when inoculated intracranially, efficient cell attachment through HS binding can contribute to viral neurovirulence. The primary aim of this review is to discuss the roles of HS binding in viral pathogenicity, including peripheral virulence and neurovirulence. Understanding how heparan sulfate functions during virus infection in vivo may prove critical for elucidating the molecular mechanism of viral pathogenesis, and may contribute to the development of therapeutics targeting HS.

Asunto(s)

Heparitina Sulfato/fisiología , Virus/patogenicidad , Humanos , Receptores Virales/fisiología , Virulencia

RESUMEN

Rab GTPases are crucial in the regulation of intracellular vesicular trafficking. A novel Rab GTPase gene, EoRab11a (GenBank accession no. EF061065), was isolated and identified from Euplotes octocarinatus cells in this study. It contains an ORF of 696-bp nucleotides, encoding 231 amino acids with a calculated molecular weight of 26.8 kDa. Alignment of EoRab11a with other Rab11 proteins from other eukaryotes demonstrated that these proteins shared 53-61% identity at the amino acid level. The recombinant EoRab11a was expressed in Escherichia coli and purified by immobilized metal chelate affinity chromatography and iron chromatography. The GTPase activity of EoRab11a was 0.0024 min(-1) detected by HPLC at 30 degrees C. Three mutations were generated at amino acids Ser21 and Gly22 positions in the G1 domain of EoRab11a. All three mutants, S21P, S21G and G22R, increased the GTPase activity in vitro. Immunofluorescence microscopy results indicated that EoRab11a was localized on the phagosomal membrane during phagocytosis of E. octocarinatus. These data show that EoRab11a possesses GTP hydrolysis activity and may participate in vesicle transport events during phagocytosis of E. octocarinatus.

Asunto(s)

Euplotes/enzimología , Euplotes/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Secuencia de Bases , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , ADN Protozoario/química , ADN Protozoario/genética , Escherichia coli/genética , Guanosina Trifosfato/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Sistemas de Lectura Abierta , Fagosomas/química , Proteínas Protozoarias/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab/química