The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21-CodonPlus (DE3)-RIL using the pGEX-4T-1 expression vector and the soluble recombinant product was purified with Glutathione Sepharose TM<'4B> by centrifugation. The soluble recombinant protein showed good immune reactions and was confirmed by Western blot using anti-CSFV-specific antibodies. Then an indirect ELISA with the purified E2 protein as the coating antigen was established to detect antibody against CSFV. The result revealed that the optimal concentration of coated antigen was 0.6 μg/well and the optimal dilution of serum was 1:80. The positive cut-off value of this ELISA assay was OD<,tested serum>/OD<,negative serum>≥2.1- The E2-ELISA method was evaluated by comparison with the indirect hemagglutination test (IHAT). When a total of 100 field serum samples were tested the sensitivity and specificity were 90.3% and 94.7% respectively. Specificity analysis showed that there were no cross-reactions between BVD serum and the purified E2 protein in the E2-ELISA.

Objective To understand the clinical features of hepatitis B virus(HBV)/hepatitis C virus(HCV)coinfected patients with different virological profiles.Methods The clinical data of 186 patients with HBV/HCV coinfection from May 1999 to May 2010 in the Third Affiliated Hospital of Sun Yat-Sen University were analyzed retrospectively.The demographic data,epidemiological data,laboratory results and pathological index were analyzed.The statistical analysis was done using t test and chi square test.Results A total of 186 patients were divided into 4 groups:66(35.5%)in HBV DNA(-)/HCV RNA(-)group,8(4.3%)in HBV DNA(+)/HCV RNA(+)group,68(36.6%)in HBV DNA(+)/HCV RNA(-)group and 44(23.7%)in HBV DNA(-)/HCV RNA(+) group.The gender composition,complication incidence,transmission among drug users,alanine aminotransferase(ALT)level,total bilirubin(TBil)level,prothrombin activity(PTA)and hapatitis B e antigen(HBeAg)negative rate were all significantly different among four groups(F or x2=11.578,8.451,11.738,2.669,5.102,4.254 and 18.413,respectively;all P<0.05).In groups of HCV RNA(-)and HCV RNA(+),the proportions of patients infected through drug abuse were 49.3%and 23.1%,respectively(x2=9.987,P:0.002)and blood transfusion transmission were 29.9%and 46.2%,respectively(x2=4.412,P=0.036).When HBV DNA was negative,the median ALT levels in HCV RNA(-)and HCV RNA(+)patients were 177 U/L and 62 U/L,respectively(t=2.200,P<0.05),median TBil levels were 133 μmol/L and 20μmol/L,respectively (t=3.608,P<0.05)and PTA were 70.6%±27.7%and 83.3%±27.8%,respectively(t=-1.982,P<0.05).The HBeAg negative rate was not affected by HCV RNA levels(t=0.707,P>0.05).The HBeAg negative rate in HBV DNA(-)patients was 85.5%,which was higher than that in HBV DNA(+)patients(59.2%)(x2=16.393,P<0.05).Conclusions HBV DNA(+/-)/HCV RNA(-)profile were major components in HBV/HCV confection.HBV DNA level is related to disease progression and prognosis,but not relate to disease severity.Liver function damage and disease severity are aggravated with HCV RNA level decreases.HBV DNA level is related to HBeAg negative rate,while HCV RNA level is not related to HBeAg seroconversion rate.

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.

The ompA gene of Chlamyia psittaci in cows was amplified by PCR with primers designed based on those reported in GenBank.The amplified ompA gene was inserted into the bacterial plasmid vector pGEX-4T-1 and then transformed into E.coli BL21(DE3) with IPTG induction. The gene was derived from plasmid pMD18-T vector and then sequenced.It was demonstrated that this recombinant fusion protein of approximately 68kD in molecular mass was highly expressed in inclusion body and more pure proteins would be produced after purification.The fusion protein specifically reacted with positive sera of bovine Chlamydia as demonstrated by Western blotting. These results indicate that this recombinant fusion protein shows good reactivity and could be used to develop the diagnostic kit for bovine Chlamydia and genetic engineering vaccine.

Background: Peste des petits ruminants (PPR), caused by the PPR virus (PPRV), is an acute and fatal contagious disease that mainly infects goats, sheep, and other artiodactyls.Peripheral blood mononuclear cells (PBMCs) are considered the primary innate immune cells. Objectives: PBMCs derived from goats were infected with PPRV and analyzed to detect the relationship between PPRV replication and apoptosis or the inflammatory response. Methods: Quantitative real-time polymerase chain reaction was used to identify PPRV replication and cytokines expression. Flow cytometry was conducted to detect apoptosis and the differentiation of CD4+ and CD8+T cells after PPRV infection. Results: PPRV stimulated the differentiation of CD4+ and CD8+ T cells. In addition, PPRV induced apoptosis in goat PBMCs. Furthermore, apoptosis and the inflammatory response induced by PPRV could be suppressed by Z-VAD-FMK and Z-YVAD-FMK, respectively.Moreover, the virus titer of PPRV was attenuated by inhibiting caspase-1-dependent apoptosis and inflammation. Conclusions This study showed that apoptosis and the inflammatory response play an essential role in PPR viral replication in vitro, providing a new mechanism related to the cell host response.

Background: Peste des petits ruminants (PPR) is an infectious disease caused by the peste des petits ruminants virus (PPRV) that mainly produces respiratory symptoms in affected animals, resulting in great losses in the world's agriculture industry every year. Singledomain variable heavy chain (VHH) antibody fragments, also referred to as nanobodies, have high expression yields and other advantages including ease of purification and high solubility. Objectives: The purpose of this study is to obtain a single-domain antibody with good reactivity and high specificity against PPRV. Methods: A VHH cDNA library was established by immunizing camels with PPRV vaccine, and the capacity and diversity of the library were examined. Four PPRV VHHs were selected, and the biological activity and antigen-binding capacity of the four VHHs were identified by western blot, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) analyses. ELISA was used to identify whether the four VHHs were specific for PPRV, and VHH neutralization tests were carried out. ELISA and western blot analyses were used to identify which PPRV protein was targeted by VHH2. Results: The PPRV cDNA library was constructed successfully. The library capacity was greater than 2.0 × 106 cfu/mL, and the inserted fragment size was approximately 400 bp to 2000 bp. The average length of the cDNA library fragment was about 1000 bp, and the recombination rate was approximately 100%. Four single-domain antibody sequences were selected, and proteins expressed in the supernatant were obtained. The four VHHs were shown to have biological activity, close affinity to PPRV, and no cross-reaction with common sheep diseases. All four VHHs had neutralization activity, and VHH2 was specific to the PPRV M protein. Conclusions The results of this preliminary research of PPRV VHHs showed that four screened VHH antibodies could be useful in future applications. This study provided new materials for inclusion in PPRV research.

Background: Peste des petits ruminants (PPR) is an infectious disease caused by the peste des petits ruminants virus (PPRV) that mainly produces respiratory symptoms in affected animals, resulting in great losses in the world's agriculture industry every year. Singledomain variable heavy chain (VHH) antibody fragments, also referred to as nanobodies, have high expression yields and other advantages including ease of purification and high solubility. Objectives: The purpose of this study is to obtain a single-domain antibody with good reactivity and high specificity against PPRV. Methods: A VHH cDNA library was established by immunizing camels with PPRV vaccine, and the capacity and diversity of the library were examined. Four PPRV VHHs were selected, and the biological activity and antigen-binding capacity of the four VHHs were identified by western blot, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) analyses. ELISA was used to identify whether the four VHHs were specific for PPRV, and VHH neutralization tests were carried out. ELISA and western blot analyses were used to identify which PPRV protein was targeted by VHH2. Results: The PPRV cDNA library was constructed successfully. The library capacity was greater than 2.0 × 106 cfu/mL, and the inserted fragment size was approximately 400 bp to 2000 bp. The average length of the cDNA library fragment was about 1000 bp, and the recombination rate was approximately 100%. Four single-domain antibody sequences were selected, and proteins expressed in the supernatant were obtained. The four VHHs were shown to have biological activity, close affinity to PPRV, and no cross-reaction with common sheep diseases. All four VHHs had neutralization activity, and VHH2 was specific to the PPRV M protein. Conclusions The results of this preliminary research of PPRV VHHs showed that four screened VHH antibodies could be useful in future applications. This study provided new materials for inclusion in PPRV research.