ABSTRACT
Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant virus-like particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21 (baby hamster kidney) cells. Collectively, the mutant virus-like particle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.
Subject(s)
Animals , Male , Cells, Cultured , Gene Expression , Genetic Vectors , Genetics , Metabolism , Green Fluorescent Proteins , Genetics , Metabolism , Immunohistochemistry , Methods , Mice, Inbred C57BL , Microscopy, Fluorescence , Methods , Neurons , Cell Biology , Metabolism , Purkinje Cells , Cell Biology , Metabolism , Semliki forest virus , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To construct a replicative anti-tumor DNA vaccine PSCK-2PFcGB based on Semliki Forest Virus (SFV) replicon vector and observe its expression in vivo and in vitro.</p><p><b>METHODS</b>The plasmid pVAX1-2PFcGB was digested with Nhe I, and the digestion product was blunted prior to further digestion with BssH II to obtain the fragment 2PFcGB, a fusion gene containing the multitarget complex antigen 2PAG encoding both the most cytotoxic T lymphocyte epitopes of human survivin and chorionic gonadotropin β chain-CTP37 of human and monkey. The 2PFcGB fragment was inserted into the PSCK vector digested by Sma I. The products with the expected size were extracted and ligated, and the positive clones were screened by kanamycin and amplified. The recombinant PSCK-2PFcGB, following identification by colony PCR and restriction endonuclease Nde I, was transfected into 293T cells via lipofectamine 2000 and its expression was detected. The recombinant plasmid was also transfected into mouse quadriceps femoris muscle to observe its expression in vivo by immunohistochemistry.</p><p><b>RESULTS</b>Nde I digestion resulted in a fragment of the expected size. Transfection with the recombinant plasmid PSCK-2PFcGB resulted in successful expression of the antigen and adjuvant molecular protein in 293T cells, with the positivity rates of 5.70% and 19.75%, respectively. The fusion tumor antigen survivin and hCGβ-CTP37 were also detected in the muscular tissues of the mice.</p><p><b>CONCLUSION</b>A novel replicative anti-tumor DNA vaccine PSCK-2PFcGB has been successfully constructed and can be expressed in 293T cells and in the muscular tissues of immunized mice, which provide a basis for further studies of the antitumor activity and immunological mechanism of the DNA vaccine.</p>
Subject(s)
Animals , Humans , Mice , Antibodies, Antinuclear , Allergy and Immunology , Cancer Vaccines , Allergy and Immunology , Gene Expression , Genetic Vectors , HEK293 Cells , Muscle, Skeletal , Metabolism , Plasmids , Semliki forest virus , Genetics , Vaccines, DNA , Allergy and ImmunologyABSTRACT
We have previously evaluated a Semliki Forest virus (SFV) replicon vectored DNA vaccine (pSFV1CS2-E2) and a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of classical swine fever virus (CSFV) in pigs. The results showed that the immunized pigs were protected from virulent challenge, but few pigs showed short-term fever and occasional pathological changes following virulent challenge. To enhance the immunogenecity of the vaccines, we tried a prime-boost vaccination strategy using a combination of prime with pSFV1CS2-E2 followed by boost with rAdV-E2. The results showed that all the immunized pigs developed high-level CSFV-specific antibodies following prime-boost immunization. When challenged with virulent CSFV, the immunized pigs (n = 5) from the heterologous boost group showed no clinical symptoms, and CSFV RNA was not detected following challenge, whereas one of five pigs from the homologous boost group developed short-term fever and CSFV RNA was detected. This demonstrates that the heterologous prime-boost vaccination regime has the potential to prevent against virulent challenge.
Subject(s)
Animals , Adenoviridae , Genetics , Metabolism , Adenovirus E2 Proteins , Genetics , Allergy and Immunology , Classical Swine Fever , Allergy and Immunology , Classical Swine Fever Virus , Genetics , Allergy and Immunology , Genetic Vectors , Immunization, Secondary , Replicon , Genetics , Semliki forest virus , Genetics , Metabolism , Swine , Vaccines, DNA , Allergy and Immunology , Viral Envelope Proteins , Genetics , Metabolism , Viral Vaccines , Allergy and ImmunologyABSTRACT
Se han utilizado los alfavirus como vectores de expresión, entre estos se encuentra el Semliki Forest virus (SFV), que es un virus envuelto, el cual, además de replicarse en el citoplasma, tiene la propiedad de expresar por separado las proteínas estructurales de las no estructurales, permitiendo un mayor control de la expresión. Los vectores derivados del SFV pueden tener una gama amplia de aplicaciones. Se pueden obtener altos títulos virales para la expresión eficiente de proteínas en diferentes líneas celulares. Pueden infectar un espectro amplio de células de mamíferos, así como de tejidos. Son prometedores para ser usados en la terapia génica como vehículos para el envío de genes específicos in vivo o in vitro, tanto en la terapia contra el cáncer como en la neuronal, especialmente cuando sólo sea necesaria una expresión a corto plazo. Sus aplicaciones en la producción de vacunas profilácticas o terapéuticas, es otro aspecto estudiado; se ha demostrado la generación de respuestas inmunes importantes contra diferentes enfermedades virales y tumorales. El desarrollo de nuevos vectores no citopáticos, de otros regulados por temperatura, así como también de otros con replicación persistente; permitirán la prolongación de la expresión. Debido a estas nuevas ventajas y a las ya conocidas, gradualmente se podrían ampliar los usos para los vectores derivados del SFV a medida que se controlen sus efectos no deseados.
Recently, Alphavirus have been used as expression vectors, among these, Semliki Forest virus (SFV), an enveloped virus, besides replicating itself in the cytoplasm, has the property to express structural proteins separately from nonstructural proteins, allowing a greater expression control. Vectors derived from SFV can have a broad range of applications. High viral titers can be obtained to efficiently express proteins in different cell lines. They can infect a wide spectrum of mammalian cells, as well as tissues. They are promising to be used on gene therapy as vehicles for specific gene delivery in vivo or in vitro, as much as in therapy against cancer as neuronal therapy, especially when a short term expression is necessary. Another studied aspect is SFV vectors applications in prophylactic or therapeutic vaccine production; the generation of important immune responses against different viral and tumor diseases is still been discussed. Development of new non-cytopathic vectors, temperature-regulated vectors, as well as others with persistent replication, will allow prolongation of expression. Due to these new advantages and to others already known, uses for vectors derived from SFV could be extended gradually, as long as undesired effects are controlled.
Subject(s)
Alphavirus , Gene Expression , Semliki forest virus , Transduction, GeneticABSTRACT
We have shown previously that a Semliki Forest virus (SFV) replicon vectored DNA vaccine (pSFV1CS-E2) expressing the E2 glycoprotein of classical swine fever virus (CSFV) conferred full protection for pigs immunized three times with 600 microg of the vaccine. This study aims to evaluate the efficacy of the DNA vaccine with lower dosage and fewer inoculations. Pigs were immunized twice with 100 microg pSFV1CS-E2 (n = 5) or control plasmid pSFV1CS (n = 3), respectively. Pigs immunized with pSFV1CS-E2 developed high titers of specific neutralizing antibodies against CSFV after the booster, and the antibody titers increased rapidly upon challenge. The immunized animals showed no clinical symptoms except short-term fever and low-level viremia, whereas the control pigs immunized with the control plasmid produced no detectable antibody before challenge and showed obvious clinical signs following challenge, and 2 pigs died on 10 or 11 days post-challenge. All control animals developed extended viremia as detected by nested RT-PCR and real-time RT-PCR. Severe pathologic lesions typical of CSFV infection were observed at necropsy. We conclude that the alphavirus replicon-vectored DNA-based vaccine can be potential marker vaccine against CSFV.
Subject(s)
Animals , Antibodies, Neutralizing , Blood , Allergy and Immunology , Antibodies, Viral , Blood , Allergy and Immunology , Body Temperature , Allergy and Immunology , Classical Swine Fever , Blood , Allergy and Immunology , Classical Swine Fever Virus , Genetics , Allergy and Immunology , Genetic Vectors , Genetics , Immunization , Plasmids , Genetics , Replicon , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Semliki forest virus , Genetics , Swine , Virology , Time Factors , Vaccines, DNA , Genetics , Allergy and Immunology , Viral Envelope Proteins , Genetics , Allergy and Immunology , Viremia , Genetics , Allergy and ImmunologyABSTRACT
The ethanolic extracts, various fractions and two pure compounds isolated from the plant N. arbortris were tested against Encephalomyocarditis Virus (EMCV) and Semliki Forest Virus (SFV). Pronounced in vitro virus inhibitory activity was observed with the ethanolic and n-butanol fractions as well as with the pure compounds arbortristoside A and arbortristoside C. In addition, ethanolic extracts and n-butanol fraction protected EMCV infected mice to the extent of 40 and 60% respectively against SFV at a daily dose of 125 mg/kg body weight.
Subject(s)
1-Butanol/administration & dosage , Administration, Oral , Alphavirus Infections/drug therapy , Animals , Cardiovirus Infections/drug therapy , Chlorocebus aethiops , Dose-Response Relationship, Drug , Encephalomyocarditis virus/drug effects , Glycosides/administration & dosage , Injections, Intraperitoneal , Iridoids/administration & dosage , Mice , Oleaceae , Phytotherapy/methods , Plant Extracts/pharmacology , Seeds , Semliki forest virus/drug effects , Vero CellsABSTRACT
DNA-based replicon expression vector pSMCTA and helper vector pSHCTA were constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with the RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3'-untranslated region (UTR). The RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter in pSMCTA and pSHCTA could drive transcription to produce replicon RNA in vivo and ex vivo. High level expression of foreign genes (GFP and LacZ) could be demonstrated by transfecting BHK21 cells with the new replicon expression vectors based on both DNA and RNA, and recombinant virus particles (RVP) be prepared by cotranfecting the expression vectors with the helper vectors. Foreign genes were also highly expressed in cells (BHK21) which were infected with RVP activated by alpha-chymotrypsin. The bifunctional replicon vectors can be used in highly efficient expression of foreign genes and preparation of RVP ex vivo, also in development of replicon vaccines and gene therapy vectors in vivo.
Subject(s)
3' Untranslated Regions , Genetics , Cloning, Molecular , DNA, Viral , Genetics , Genetic Vectors , Genetics , RNA, Viral , Genetics , Recombinant Proteins , Genetics , Replicon , Genetics , Semliki forest virus , Genetics , Virion , Genetics , Virus Assembly , GeneticsABSTRACT
En éste trabajo se presentan los mecanismos y las moléculas que participan de la apoptosis en células de mamífero. Se discute la función de la mitocondria, se relacionan los distintos controles del sistema con patologías humanas y se presentan algunos virus neurotrópicos donde existe una importante conexión con la apoptosis. Asimismo, se indican algunos factores participantes del proceso que tienen una veta promisoria en el tratamiento de enfermedades donde la desregulación de la muerte celular programada es la causa de la patología en cuestión
Subject(s)
Humans , Apoptosis , Caspases , Genes, bcl-2 , Receptors, Tumor Necrosis Factor , Alphavirus , Alphavirus Infections , Biotechnology , Bunyaviridae Infections , Caspases , Flavivirus , Flavivirus Infections , Genetic Therapy , Neoplasms , Neurodegenerative Diseases , Neurons , Oligonucleotides, Antisense/therapeutic use , Orthobunyavirus , Reoviridae , Reoviridae Infections , Semliki forest virusABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of Lead (Pb) acetate exposure on Semliki forest virus (SFV) pathogenesis in mice.</p><p><b>METHODS</b>Different doses (62.5, 125, 250 and 500 mg/Kg body weight) of Pb dissolved in normal saline were given to mice by oral intubation in a sub-acute (28 days) and sub-chronic (90 days) regimen followed by SFV infection. Morbidity, mortality, clinical symptoms, mean survival time (MST), changes in body and organ weight, accumulation of lead in soft tissues, virus titre in brain and histopathological alterations were compared between lead exposed and infected groups.</p><p><b>RESULTS</b>Early appearance of virus symptoms, increased mortality, decreased MST, enhanced SFV titre and greater tissue damage were observed in lead exposed-SFV-infected mice.</p><p><b>CONCLUSION</b>Pre-exposure to lead increases the susceptibility of mice towards SFV infection. Further studies are suggested in view of the persistence of lead in the environment and the possibility of infection by microbial pathogens.</p>
Subject(s)
Animals , Mice , Alphavirus Infections , Brain , Pathology , Dose-Response Relationship, Drug , Kidney , Pathology , Lead , Toxicity , Liver , Pathology , Semliki forest virus , VirulenceABSTRACT
Angiogenesis is recognized as a critical factor in the growth of tumor cells and plays a key role in the tumor metastasis. Recent studies for antiangiogenic substances are getting popular. The angiostatin, one of the antiangiogenic substances, leads to the increased apoptosis of the tumor cells by inhibiting the neovascularization of the tumor. The angiostatin was identified as the internal fragments of the plasminogen which has no antiangiogenic activity. By hydrolysis of the plasminogen, the angiostatin can be produced. In this study, we constructed the SFV-derived DNA vector by employing the cytomegalovirus immediate early enhancer/ promoter (CMV). This vector makes it possible to transfect the cells with DNA without the in vitro transcription process. The C-myc epitope and polyhistidine residue sequences were placed in downstream of the angiostatin gene to make it eligible to detect the expressed protein. The murine Ig kappa-chain V-J2-C signal sequence was placed in upstream to secrete the expressed protein from the cells. We confirmed the expression of angiostatin in the BHK-21 cells using DNA-based SFV replicon.
Subject(s)
Animals , Cricetinae , Angiostatins/analysis , Base Sequence , Cell Line , DNA Primers , Gene Expression Regulation, Viral , Immunohistochemistry , Kidney , Plasmids , Replicon/genetics , Semliki forest virus/genetics , TransfectionABSTRACT
The ORF5 gene encodes a major envelope glycoprotein (GP5), which is one of the three major proteins of porcine reproductive and respiratory syndrome virus (PRRSV). The GP5 protein has been known to be a 24.5-26kDa N-glycosylated envelope protein. The GP5 is involved in inducing neutralizing antibodies. For this reason, the GP5 is primary candidate for the PRRSV subunit vaccine. To produce the native form of GP5 in mammalian cells, we have cloned the ORF5 gene from PRRSV CNV-1 into the Semliki Forest virus (SFV)-based expression vector, resulting in recombinant pSFV-ORF5. By the infection with recombinant pSFV-ORF5 to BHK-21 cells, the GP5 expression was confirmed by immunocytochemistry and immunoblotting assay. The recombinant virus particle harboring ORF5 gene was infectious to BHK-21 and MARC-145. The RNA synthesis and expression of GP5 in the infected cell was also confirmed by RT-PCR.