The gene encoding the nucleocapsid (N) protein of vesicular stomatitis virus (VSV-NJ) was subcloned from pMD-VN5, and inserted into pBAD/Thio TOPO vector. The recombinant plasmid was identified by restriction analysis and PCR. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted N gene. The results of SDS-PAGE and Western immunoblotting revealed that the N protein was expressed in Escherichia coli LGM194 in a high level and the recombinant fusion protein, which contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. It had a molecular mass of approximately 63.5 kD and immunologically reactive activity. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of vesicular stomatitis using 186 serum samples from experimentally infected goats and guinea-pigs with VSV-NJ and VSV-IN, and from field origin and reference serum samples. The sensitivity and specificity of the ELISA were compared with those of the standard microtiter serum neutralization (MTSN) tests. The ELISA and MTSN test results were highly correlated for detection of VSV antibodies. The ELISA was as sensitive as the SN assay in detecting positive serum to VSV. The correlation between SN titers and ELISA titers was statistically significant. These data suggest that the recombinant fusion N protein of VSV could be used as a recombinant test antigen for the serodiagnosis of vesicular stomatitis. The ELISA based on the reconmbinant nucleocapsid protein may offer the best combination of rapidity, sensitivity, simplicity, economy, and laboratory biosafety of any of the methods yet developed for VSV serodiagnosis. This study lay on foundation for the development of the diagnosis methods in serology for VSV.
Amino Acid Sequence ; Animals ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Molecular Sequence Data ; Neutralization Tests ; Nucleocapsid Proteins ; chemistry ; genetics ; immunology ; isolation & purification ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Serologic Tests ; Vesicular stomatitis Indiana virus ; genetics ; Vesicular stomatitis New Jersey virus ; genetics

OBJECTIVE: To study the result of HNE-1 cancer cells lesion and HNE-1 cancer cells apoptosis caused by vesicular stomatitis virus in vitro. METHOD: Firstly, HNE-1 cancer cells of human nasopharyngeal carcinoma were cultured in vitro. Secondly, tumor cells were treated with vesicular stomatitis virus (VSV) in different concentration (0.1 moi, 1.0 moi, 10.0 moi and 100.0 moi), while cells without treatment were used as blank assay. Finally, we observed the lesion of HNE-1 cells treated with VSV for 24 to 48 hours under invert microscope, compared with HNE-1 cells of blank assay. By measurement of MTT reduction assay and Hoechst 33258- staining under fluorescence microscope, the effect of VSV killing HNE-1 cancer cells and the induction of apoptosis by VSV in HNE-1 cancer cells were investigated in vitro. RESULT: Compared with blank assay, HNE-1 cancer cells of human nasopharyngeal carcinoma treated with VSV showed phenomenon of lesion under invert microscope. Followed the increase of concentration of VSV, cell survival rate of HNE-1 cancer cells decreased. The research methods of MTT reduction assay and Hoechst 33258 staining under fluorescence microscope defined the effect of VSV killing HNE-1 cancer cells and confirmed that VSV inducing apoptosis of HNE-1 cancer cells in vitro. CONCLUSION VSV not only exhibited the anti-tumor activity in HNE-1 cancer cells of human nasopharyngeal carcinoma but also showed the potential power of inducing apoptosis of HNE-1 cancer cells in vitro.
Apoptosis ; Cell Line, Tumor ; Cell Survival ; Humans ; Nasopharyngeal Neoplasms ; pathology ; Oncolytic Virotherapy ; Vesiculovirus

OBJECTIVE: To investigate apoptosis induced by vesicular stomatitis virus (VSV) in HNE-1 cancer cells of human nasopharyngeal carcinoma by models of nude mice-BALB/c in vivo. METHOD: HNE-1 cells are collected from culture bottle and infected infra right-side post-back epithelium of nude mice-BALB/c (6 x 10(6) cells/0.1 ml/each mice) to create HNE-1 tumor models of nude mice-BALB/c. When the diameter of HNE-1 tumors is 5 to 8 millimeter, HNE-1 tumor models are treated with VSV (1 x 10(8) pfu /ml) or Saline. By Hoechst 33258-staining under fluorescence microscope, induction of apoptosis by VSV in HNE-1 tumor models are recorded and studied, compared with that by Saline in HNE-1 tumor models in vivo. RESULT: Compared with control group of saline, apoptosis of HNE-1 cancer cells of human nasopharyngeal carcinoma increase apparently in the remaining tumor cells of nude mice treated by VSV (P < 0.01). CONCLUSION The present study suggests that the treatment with VSV could augment the apoptosis cells of human nasopharyngeal carcinoma in vivo.
Animals ; Apoptosis ; Cell Line, Tumor ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nasopharyngeal Neoplasms ; Neoplasm Transplantation ; Oncolytic Virotherapy ; Vesiculovirus ; Xenograft Model Antitumor Assays

In the nationwide epidemiological investigation, SVCV-741 was for the first time isolated in Beijing region, China in 2003, and designated as SVCV Asian strain. In this paper, we compared SVCV-741 (Asian strains isolated in China) with SVCV-10/3 (Europe reference strain) on their physico-chemical, biological and morphological characteristics. The results indicated that there were no distinct differences between two SVCV strains on phycico-chemical and morphological characteristics. The main existing differences were: (1) The stability of SVCV-741 to temperature in cell culture was higher than that of SVCV-10/3, which might have some evolutionary and biological implication of SVCV; (2) No SVC outbreak ever occurred caused by SVCV-741;Furthermore we found that both SVCV-741 and SVCV-10/3 grew faster and produced higher virus titer in CO cells than other cell lines. It indicated that CO cell lines might be useful tool for SVCV research.
Animals ; Carps ; virology ; Cell Line ; China ; Europe ; Fish Diseases ; virology ; Fishes ; Microscopy, Electron, Transmission ; Reverse Transcriptase Polymerase Chain Reaction ; Rhabdoviridae Infections ; veterinary ; virology ; Vesiculovirus ; genetics ; growth & development ; isolation & purification ; ultrastructure

In order to ensure the biosafety of the IFN-gamma antiviral activity assay, we used a replication-deficient VSV carrying GFP as an interferon sensitive indicator virus (VSVdeltaG*G). The antiviral activities of porcine IFN-gamma expressed in Escherichia coli and in baculovirus on MDBK cells were assessed. The results showed that the antiviral activity of porcine IFN-gamma expressed in baculovirus could reach 10(5) IU/mL, while the porcine IFN-gamma expressed in E. coli showed some antiviral activity (32 IU/mL) after refolding. The results of the VSVdeltaG*G-based antiviral assay were almost identical to that of the VSV*GFP-based assay, suggesting it is highly feasible to use VSVdeltaG*G as a substitute for VSV*GFP, making assays for IFN-gamma antiviral activity safer and more accurate.
Animals ; Antiviral Agents ; pharmacology ; Baculoviridae ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Interferon-gamma ; biosynthesis ; genetics ; metabolism ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Swine ; Vesiculovirus ; drug effects ; physiology

The full-length porcine interferon gamma(PoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBac -PoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-gamma. Recombinant baculovirus, rBac-PoIFN-gamma, was generated for expressing PoIFN-gamma, by transfecting rBacmid-PoIFN-gamma with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-gamma in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-gamma expressed by the rBac-PoIFN-gamma can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-gamma can be specifically blocked by anti-PoIFN-gamma mouse serum. The antiviral titer of culture supernatant of insect cells infected by rBac-PoIFN-gamma is 2 x 10(4) IU/mL. The results demonstrat that the rBac-PoIFN-gamma can express rPoIFN-gamma efficiently and rPoIFN-gamma has high antiviral activity.
Animals ; Antiviral Agents ; metabolism ; pharmacology ; Baculoviridae ; genetics ; Blotting, Western ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Gene Expression ; Green Fluorescent Proteins ; genetics ; metabolism ; Immune Sera ; immunology ; Interferon-gamma ; genetics ; immunology ; metabolism ; pharmacology ; Mice ; Mice, Inbred BALB C ; Microscopy, Fluorescence ; Recombinant Proteins ; Spodoptera ; Swine ; Vesiculovirus ; genetics ; Virus Replication ; drug effects

Transgenic chicken and oviduct bioreactor are growing to be one of the hotspot of scientific study in the field of biology. The most successful method of producing transgenic chicken is pseudotyped retrovirus vector system, but no one has reported the production of transgenic chicken by retrovirus system recently in our country. In order to accelerate our study in this field, we introduced the relevant technical methods such as packaging retrovirus and vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped retrovirus, optimizing the conditions of packaging retrovirus, concentrating VSV-G pseudotyped retrovirus, helper virus assays, and microinjection of retrovirus. Furthermore, we successfully conducted in vivo study for detecting the marker gene EGFP of chicken embryo as well as in vitro study for detecting that gene of chicken embryo myoblast (CFM), thus we have provided an applied technical platform for studies of transgenic chicken in the future.
Animals ; Animals, Genetically Modified ; Chick Embryo ; Chickens ; genetics ; DNA Primers ; Genetic Vectors ; genetics ; Retroviridae ; genetics ; Vesicular stomatitis Indiana virus ; genetics

To determine whether addition of vitamin C (Vit C) to single-unit plasma could influence the efficacy of inactivating viruses and could maintain the activity of plasma proteins by methylene blue (MB)-light treatment. Vesicular stomatitis virus (VSV) Indiana strain was used as the indicating virus. Human plasma containing VSV was added with different concentrations of Vit C and final concentration 1 micromol/L MB and irradiated by fluorescence at an intensity of 40,000 lx, samples were collected at different times for detection. Cytopathic effect was used to test the effect of virus inactivation. A segment of the nucleic acid encoding capsid protein of VSV was amplified with RT-PCR. Some methods, such as the Clauss method, the one-stage method, microimmunoelectrophoresis, were used to investigate the changes of plasma components. The results showed that when the VSV plasma was added with 240 micromol/L Vit C and treated by MB-light irradiation for 60 min, the titer of VSV decreased by more than 8 lg TICD50/ml. Meanwhile, target segment amplification of VSV was also negative. The recovery rates of fibrinogen and coagulation factor VIII (FVIII: C) were 83.55% and 81.67% respectively, which had significant difference comparing with the routine MB-fluorescent light treatment. Most of plasma proteins were not affected significantly. No change in immunogenicity of these proteins was observed by using microimmunoelectrophoresis. It is concluded that virus inactivation is not influenced and plasma proteins are effectively protected by Vit C. Vit C can be used as a protector and is beneficial to improving the quality of plasma subjected to MB- photodynamic treatment.
Ascorbic Acid ; pharmacology ; Blood Proteins ; metabolism ; Humans ; Light ; Methylene Blue ; pharmacology ; Plasma ; virology ; Vesicular stomatitis Indiana virus ; drug effects ; Virus Inactivation ; drug effects

In the last decade, we have gained significant understanding of the mechanism by which vesicular stomatitis virus (VSV) specifically kills cancer cells. Dysregulation of translation and defective innate immunity are both thought to contribute to VSV oncolysis. Safety and efficacy are important objectives to consider in evaluating VSV as a therapy for malignant disease. Ongoing efforts may enable VSV virotherapy to be considered in the near future to treat drug-resistant ovarian cancer when other options have been exhausted. In this article, we review the development of VSV as a potential therapeutic approach for recurrent or drug-resistant ovarian cancer.
Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Humans ; Neoplasm Recurrence, Local ; Oncolytic Virotherapy ; methods ; Ovarian Neoplasms ; pathology ; therapy ; virology ; Vesicular stomatitis Indiana virus ; physiology ; Virus Replication

To clone porcine bone marrow stromal antigen-2 (BST-2) gene, construct its recombinant eukaryotic expression plasmid and induce the expression of the fusion antiviral protein, we amplified BST-2 gene by RT-PCR from the total RNA extracted from PK15 cells. The recombinant expression plasmid pcDNA-BST-2 was constructed and then was transfected into HEK293T cells to expresse the BST-2 fusion protein. Western blot and indirect immunofluorescence assay (IFA) were performed, and the biological activity was detected. The results showed that the construction of recombinant plasmid pcDNA-BST-2 was confirmed by restriction enzyme digestion and sequencing. The expressed product had antiviral activity against Vesicular stomatitis virus (VSV), Avian influenza virus (AIV) and Porcine reproductive and respiratory syndrome virus (PRRSV). In conclusion, the research paves the way for further research on bioactivity assayand antiviral medication.
Animals ; Antigens, CD ; genetics ; immunology ; Cell Line ; Chickens ; Cloning, Molecular ; Gene Expression ; Humans ; Influenza in Birds ; immunology ; virology ; Orthomyxoviridae ; physiology ; Porcine Reproductive and Respiratory Syndrome ; immunology ; virology ; Porcine respiratory and reproductive syndrome virus ; physiology ; Swine ; Vesicular Stomatitis ; immunology ; virology ; Vesicular stomatitis Indiana virus ; physiology ; Virus Replication