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.

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.

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.

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) 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.

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.

Objective To investigate short-term efficacy and security of transplantation of human umbilical cord mesenchymal stem cells (HUCMSCs)in the course of treatment of decompensated cirrhosis.Methods Ninety-six Hepatitis B patients with decompensated cirrhosis were enrolled,among which 50 were sbject to transplantation of HUCMSCs in addition to routine therapy (the treatment group) and the rest patients were underwent routine therapy(the control group).We observed the efficacy and security at 2,4,6 and 12 week of the treatment course.Results The symptoms of atigue,anorexia,bloating and edema were greatly relieved in both groups after 4 weeks,and sustained remission after 4-12 weeks.The overall survival rate was 96％ after 12 weeks of the treatment group,2 patients were died after 8 and 12 weeks of the HUCMSCs transplantation due to hepatic encephalopathy repecticely.Compared to the baseline,ALT,AST,TBil and ALB(albumin) were improved after 2,4,6 and 12 weeks in both groups(P ＜ 0.05),and the averages of ALB(albumin) of treatment group were higher than the control group.PT(prothrombin time) and PTA(prothrombin time activity) were improved after 4,6,12 weeks in the treatment group (P ＜ 0.05),however,there were no differences in the control group during the 4,6 and 12 weeks(P ＞0.05).During the course of HUCMSCs tranplantation,the security was satisfied and no special side effects were observed.Conclusion The transplantation of HUCMSCs for therapy of Hepatitis B patients with decompensated cirrhosis is a safety and effective therapy,and can improved ALB,PT,PTA,liver function and clinical symptoms within a short period,which is an alternative method of treatment recommended.

Objective To investigate short-term efficacy and security of telbivudine as a sequential therapy in the HBeAg positive chronic hepatitis B (CHB) patients with pegylated IFNα-2a treatment failure.Methods 27 CHB patients with HBeAg positive and HBV DNA detectable after a 48-week pegylated IFNα-2a therapy were enrolled into this study,and were assigned to group A,with telbivudine as a sequential therapy.54 CHB patients with HBeAg positive were assigned to group B,with telbivudine as a naive treatment.To assessment the efficacy and security of telbivudine at week 48.Results At week 12,the rates of aminotransferases normalization,HBeAg seroconversion and HBV DNA undetectable (＜ 100 IU/ ml) among the two groups were 59.3％,14.8％,66.7％ vs 75.9％,5.6％,46.3％ respectively.At week 24,the rates among the two groups were 92.6％,25.9％,70.3％ vs 92.6％,7.4％,85.2％,respectively.At week 48,the rates among the two groups were 88.9％,29.6％,81.5％ vs 98.1％,28.0％,83.3％ respectively.All these were not statistically significant.But the rate of HBeAg seroconversion in group A is higher than that in group B.The virological breakthrough rate of group B at week 48 is 14.8％,no virological breakthrough was observed in group A.During the treatment,38.3％ of patients had creatine kinase(CK) elevation,but there was no case stopping treatment for severe adverse effect.Conclusion CHB patients with HBeAg positive after a 48-week pegylated IFNα-2a treatment failure can also achieve the same efficacy and security as the na(i)ve treatment,after receiving telbivudine as a sequential therapy.

Objective To investigate the efficacy of Telbivudine as sequential therapy for HBeAg positive CHB patients with pegylated IFNα-2a (PEG-IFNα-2a)treatment failure.Methods After a 48-week pegylated IFNα-2a therapy,102 HBeAg positive CHB patients,whose HBeAg expression was positive and HBV DNA load was Detectable,were enrolled.These patients were randomly divided into two groups.Group A,included 52 patients,were treated with Telbivudine as a sequential therapy after a 3 months or longer wash-out period.Group B,included 49 patients,were treated with Telbivudine as a sequential therapy without wash-out period.The rate of ALT normalization,HBeAg seroconversion,the negative rate of HBV DNA and the level of creatine kinase in the two groups were observed and compared,respectively.Results HBeAg seroconversion of Group A at 3,6,12 months was 7.69％,15.3％,21.1％,respectively.HBeAg seroconversion undetectable of Group B at 3,6,12 months was 22.4％,32.6％,38.8％,respectively.Furthermore,there was statistically significant difference in HBeAg seroconversion between the two groups (P ＜ 0.05).However,there were not statistical difference in the rate of ALT normalization,the negative rate of HBV DNA and the level of creatine kinase between the two groups at 3,6,12 months.Conclusion Telbivudine,which was used as a sequential therapy without wash-out period for HBeAg positive CHB patients after PEG-IFNα-2a treatment failure,was secure.Moreover,HBeAg seroconversion of patients treated with Telbivudine as a sequential therapy without wash-out period was higher than those with wash-out period.