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.

After sequencing, we amplified and cloned foot-and-mouth disease virus (FMDV) O/QYYS/s/06 whole genome by three fragments. These three fragments were cloned into vector P43 one by one to construct recombinant plasmid P43C, which carried the full-length cDNA of FMDV O/QYYS/s/06. Then, plasmid P43C and plasmid T7 expressing T7 RNA polymerase were co-transfected into BHK-21 cells. After 48 h, we harvested the culture broth from transfected BHK-21 cells and inoculated into 2-3 day-old sucking mice. After four generation passage, the virus harvested from sucking mice was confirmed to be type O FMDV by the indirect hemagglutination test, sucking mice's neutralization test and sequencing. The results showed that we have successfully constructed the full-length cDNA clone of FMDV O/QYYS/s/06 strain.
Animals ; Animals, Newborn ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Viral ; biosynthesis ; genetics ; Foot-and-Mouth Disease ; virology ; Foot-and-Mouth Disease Virus ; classification ; genetics ; pathogenicity ; Mice ; Transcription, Genetic ; Transfection

Recombinant structural protein VP1 of foot-and-mouth disease virus serotype O was expressed in Escherichia coli and then purified using Nickel affinity chromatography. A chemiluminescent enzyme immunoassay (CLEIA) method was established using the purified recombinant protein as coating antigen to detect antibody of foot-and-mouth disease virus serotype O in swine. The specificity of VP1-CLEIA method is 100%. The coefficients of variation in the plate and between plates are 1.10%-6.70% and 0.66%-4.80%, respectively. Comparing with the commercial indirect ELISA kit or liquid phase block ELISA kit, the calculated coincidence rate is 93.50% or 94.00%. The high specificity and stability suggested this detection method can be used to monitor the antibody level of foot-and-mouth disease virus serotype O in swine.

The aim of this study is to quantify the 146S antigen in foot-and-mouth disease virus (FMDV) inactivated vaccine by size-exclusion chromatography (SEC). The analysis was performed on a TSKgel G4000SWXL column (7.8 mm×30 cm), with a pH 7.2 buffer salt system as the mobile phase. The flow rate was 0.6 mL/min, the injection volume was 100 μL and the detection wavelength was 259 nm. The calibration curve was established by using purified inactivated FMDV (serotype O) 146S antigen; 3 batches of vaccine formulated by inactivated antigen solution were tested to verify the accuracy, reproducibility, specificity and tolerability of the method. At last 16 batches of vaccine were determined by the SEC method. Results showed a good linearity between peak area and concentration of 146S antigen in the range between 0.56 and 67.42 μg/mL (R2=0.996, n=10), and the average recovery rate of 146S antigen in the 3 batches of vaccine formulated in lab were 93.6% (RSD=2.7%, n=3), 102.3% (RSD=2.6%, n=3), and 95.5% (RSD=5.1%, n=3). The method was proved accurate and reliable with good reproducibility (RSD=0.5%, n=6), and applied to determine 16 batches of the commercial FMDV vaccine. According to the above results, the SEC method is high effective for 146S antigen quantify in the inactivated FMDV vaccine and would provide strong support for the vaccine quality control.

The HA gene of H9N2 influenza virus (A/chicken/Hunan/04.14 (H9N2)) was amplified and sequenced. The RNA was synthesized by in vitro transcription. The RNA transcription solutions were diluted to 10⁹ copies/μL using the RNA storage solution. The aliquoted RNA solutions were used to evaluate the homogeneity and stability. The results were determined by the average value obtained from four independent laboratories. Furthermore, the fluorescence quantitative RT-PCR method was also developed to verify the detection accuracy of clinical samples. The detection limit of this method is approximately 10 copies. Taken together, the RNA transcription solution established in our study can used as positive standard reference for rapid detection of H9N2 influenza virus.

We developed a pre-treatment method to remove interfering substances during quantification of 146S antigens in foot-and-mouth disease (FMD) vaccines by high performance size exclusion chromatography (HPSEC). Three methods, including ultracentrifugation, PEG precipitation and nuclease digestion, were optimized and compared for removal efficiency of the interfering impurities in FMD vaccines. Under optimized conditions, the 146S contents in two batches of FMD vaccines were determined to be 7.1 and 7.6 μg/mL by ultracentrifugation, 9.7 and 10.4 μg/mL by PEG precipitation, and 10.5 and 10.4 μg/mL by nuclease digestion. The optimal condition for nuclease digestion using Benzonase determined by response surface method was as follows: appending Benzonase into 200 μL of antigen phase to a final concentration of 421 U/mL and incubating at 25.1 °C for 1.29 h. This method has advantages including efficient removal of the interfering impurities, fast processing speed, and mild operating conditions. Then 12 bathes of FMD vaccines with different serotypes produced by 4 manufacturers were tested to verify the established treatment method. Results showed the method was applicable to various FMD vaccines with good reproducibility (RSD<5.3%, n=3). The developed method removed interference from impurities during quantification of 146S, and therefore would broaden the application of HPSEC in vaccine quality control and ensure the accuracy and reliability.
Animals ; Chromatography, Gel ; Foot-and-Mouth Disease ; Foot-and-Mouth Disease Virus ; Reproducibility of Results ; Viral Vaccines

This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×10⁶ (±4.34%) Da and 2.40×10⁶ (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×10⁶ (±2.94%) Da and 2.88×10⁶ (±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×10⁶ (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all < 1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R² of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.
Animals ; Antibodies, Viral ; Capsid Proteins ; Chromatography, Gel ; Circoviridae Infections/prevention & control* ; Circovirus ; Lasers ; Swine ; Vaccines, Inactivated ; Vaccines, Virus-Like Particle ; Viral Vaccines