Venezuelan equine encephalitis (VEE) is a zoonotic disease caused by the Venezuelan equine encephalitis virus (VEEV) complex. This disease has not yet been reported in China, and it is therefore essential to establish a rapid and accurate method for detection of the virus in order to prevent and control this disease. In this study, a one-step real-time quantitative RT-PCR method was developed for the detection of the VEEV complex. A pair of specific primers and a Taqman probe were designed corresponding to a conserved region of the VEEV gene nspl, allowing the detection of all known strains of different sub- types of the virus. Using RNA synthesized by in vitro transcription as template, the sensitivity of this method was measured at 3.27 x 10(2) copies/microL. No signal was generated in response to RNA from Chikungunya virus (CHIKV), nor to RNA encoding the nsp1 fragment of Eastern equine encephalitis virus (EE-EV) or Western equine encephalitis virus (WEEV), all of which belong to the same genus as VEEV. This indicates that the method has excellent specificity. These results show that this one-step real-time quantitative RT-PCR method may provide an effective tool for the detection of VEEV in China.
China ; DNA Primers ; genetics ; Encephalitis Virus, Venezuelan Equine ; classification ; genetics ; isolation & purification ; Encephalomyelitis, Venezuelan Equine ; virology ; Humans ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods

Objective To design a new type of hybrid bioartificial liver (HBAL), evaluate its efficacy in vitro, and explore the feasibility in clinical application.Methods CL-1 human hepatocytes were cultured on microcarriers for 5 days, when cell count reached about 4.0 × 109 with cell density of about 4.0 × 107/ml.CL-1 cells cultured on microcarriers in home-made bioreactor constitute the biological part of the HBAL.The abiotic part included blood perfusion and bilirubin adsorption, and blood pump was employed as the circulation driver, which were parts of HBAL.The changes of the concentrations of indirect bilirubin (UBD), chenodeoxycholic acid (CDCD), cholic acid (CA), blood ammonia (AA), AST, ALT and LDH were observed under the condition of in vitro circulation.Meanwhile, the function, morphology and the cell activity of CL-1 cells were also observed.Results After in vitro circulation for 24 h, the concentrations of UBD, CDCD, CA and AA significantly decreased from (335.3 ± 6.0) μmol/L, (395.0 ± 5.6) μmol/L, (155.7 ± 4.5) μmol/L, (39.0 ± 2.6) μmol/L at 0 h to (106.0 ± 10.9) μmol/L, (131.8 ± 28.7) μmol/L, (42.2 ± 7.3) μmol/L, (3.5 ± 1.0) μmol/L, respectively.At 48 h, ALT, AST and LDH significantly increased from (25.9 ± 4.2) IU/L, (22.0 ± 3.6) IU/L, (0.28 ± 0.09) μmol/L to (31.0 ± 2.6) IU/L,(31.6 ± 8.0) IU/L, (0.41 ± 0.12) μmol/L, meanwhile the count and vitality of CL-1 cells were significant declined.Conclusions (1) In the new HBAL system, CL-1 cells can keep its viability and function in vitro;and (2) the HBAL appears to be effective in purifying the serum in liver failure simulation model by clearing out non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride, which seems to be a promising therapeutic option.