ABSTRACT
Classical swine fever virus (CSFV), the pathogen of classical swine fever (CSF), causes severe hemorrhagic fever and vascular necrosis in domestic pigs and wild boar. A large number of evidence has proven that non-structural 5A (NS5A) is not only a very important part of viral replication complex, but also can regulate host cell’s function; however, the underlying mechanisms remain poorly understood. In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A’s function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database revealed 16 interactive proteins: DDX5, PSMC3, NAV1, PHF5A, GNB2L1, CSDE1, HSPA8, BRMS1, PPP2R3C, AIP, TMED10, POLR1C, TMEM70, METAP2, CHORDC1 and COPS6. These proteins are mostly related to gene transcription, protein folding, protein degradation and metabolism. The interactions detected by the Y2H system should be considered as preliminary results. Since identifying novel pathways and host targets, which play essential roles during infection, may provide potential targets for therapeutic development. The finding of proteins obtained from the SUVEC cDNA library that interact with the CSFV NS5A protein provide valuable information for better understanding the interactions between this viral protein and the host target proteins.
ABSTRACT
A simple and rapid assay for the detection of Classical swine fever virus(CSFV)was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP).This study describes the amplification of the genomic RNA of CSFV under isothermal conditions(63℃)within one hour,using a set of six primers(two outer primers,two inner primers and two loop primers).This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR.This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV.PRRSV.SIV.PRV-PCV,thus showed a good specificity.Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition,either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye.Because RT-LAMP is low-cost and produces rapid results,it has the potential to be an excellent tool for CSFV surveillance in the field,especially in developing countries.
ABSTRACT
In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both ≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.
ABSTRACT
E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.