Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

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

Equine interferon-gamma (eIFN-gamma) expressed both in E. coli and baculovirus were evaluated for antiviral activity against recombinant Vesicular Stomatits Virus expressing green fluorescence protein (rVSV-GFP) in EFK-78 cells. The assays were conducted in 96-well plate. Virus infectivity was measured by quantifying GFP-positive cells, instead of quantifying the CPE reduction. Prior to infection of EFK-78 cells with rVSV-GFP, the cells were incubated with eIFN-gamma. The GFP expression in the EFK-78 cells dramatically decreased in the cells treated with eIFN-gamma in a dose-dependent manner, comparing with the mock-treated cells. The titers of antiviral activity were 1 x 10(3) AU/mL and 1 x 10(5) AU/mL of eIFN-gamma expressed from E. coli and baculovirus, respectively. The antiviral activities of the recombinant eIFN-gamma were highly efficient and specific, as it was blocked by mAbs against eIFN-gamma.
Animals ; Antibodies, Monoclonal ; immunology ; Antiviral Agents ; metabolism ; pharmacology ; Baculoviridae ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; metabolism ; Horses ; Interferon-gamma ; biosynthesis ; genetics ; pharmacology ; Recombinant Proteins ; Vesicular stomatitis Indiana virus ; drug effects ; metabolism

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