Jatrolignans C and D: New Neolignan Epimers from Jatropha curcas.

Two new neolignans jatrolignans, C (1) and D (2), a pair of epimers, were isolated from the whole plants of Jatropha curcas L. (Euphorbiaceae). Their structures were determined with HRESIMS, IR, and NMR data analysis, and electronic circular dichroism (ECD) experiments via a comparison of the experimental and the calculated ECD spectra. Their antichlamydial activity was evaluated in Chlamydia abortus. They both showed dose-dependent antichlamydial effects. Significant growth inhibitory effects were observed at a minimum concentration of 40 µM.

Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses.

Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and evaluated subsequently for its effectiveness in detecting simultaneously single and mixed infections in swine. Specific primers for three DNA viruses and three RNA viruses, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive detecting at least 450pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty clinical samples and aborted fetuses collected from 4- to 12-week-old piglets were detected among 39 samples tested by both uniplex and multiplex PCR, showing highly identification. Because of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in swine.

Oral vaccination with attenuated Salmonella enterica serovar Typhimurium expressing Cap protein of PCV2 and its immunogenicity in mouse and swine models.

Attenuated Salmonella enterica serovar Typhimurium (S. typhimurium) was selected as a transgenic vehicle for the development of oral vaccines against Porcine circovirus type 2 (PCV2). The Cap-encoding gene of PCV2 was amplified by PCR and cloned into expression vector pYA3341. The recombinant plasmid pYA3341-Cap was transformed into attenuated S. typhimurium X4550. BALB/c mice were inoculated orally with various doses of attenuated S. typhimurium X4550/pYA3341-Cap. The bacterium was safe to mice at dose of 2×10(9)cfu and eventually eliminated in the spleen and mesenteric lymph nodes at 4 weeks post-immunization. The flow cytometry analysis showed that the percentage of CD4(+) T cells and CD4(+)/CD8(+) ratio were increased significantly in mice immunized with attenuated S. typhimurium X4550/pYA3341-Cap. Vaccine tests in swine showed that the oral immunization with attenuated S. typhimurium X4550/pYA3341-Cap could elicit significantly higher Cap antibody titers in the treated swine than the control groups. Virus neutralization test showed that serum from the swine treated with attenuated S. typhimurium X4550/pYA3341-Cap had significant levels of neutralization activities. The swine lymphocyte proliferative responses indicated that attenuated S. typhimurium X4550/pYA3341-Cap could induce obvious cellular immune response. An in vivo challenge study showed the swine treated with attenuated S. typhimurium X4550/pYA3341-Cap had significantly lower PCV2-associated lesions and PCV2 viremia than the control groups. The results indicated that attenuated S. typhimurium X4550/pYA3341-Cap can be a potential vaccine against PCV2 infections.

Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.

The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections.

Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection.

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections.

Baculovirus surface display of E envelope glycoprotein of Japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models.

Japanese encephalitis virus (JEV), an important pathogen in humans and animals, is capable of causing febrile syndrome, encephalitis and death. The E glycoprotein of JEV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. In this work, we have succeeded in construction of one recombinant baculovirus BacSC-E expressing His6-tagged E with the baculovirus envelope protein gp64 TM and CTD. After infection, E was expressed and anchored on the plasma membrane of Sf-9 cells, as demonstrated by Western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the E glycoprotein was successfully displayed on the viral surface. Vaccination of mouse and swine with recombinant baculovirus BacSC-E successfully induced neutralizing antibody response and protective immunity toward a lethal challenge of the JEV. Taken all findings together, our results indicate that the recombinant baculovirus BacSC-E can be a potential vaccine against JEV infections. This finding provides valuable information for establishing subunit vaccines for JEV antigenic complex viruses. This is a fresh research demonstrating the potential of E-pseudotyped baculovirus as a JEV vaccine.