Baculovirus surface display of E(rns) envelope glycoprotein of classical swine fever virus.

Classical swine fever virus (CSFV) causes significant losses in the pig industry in many countries. E(rns) is an envelope glycoprotein of CSFV which is known to induce virus-neutralizing antibodies and protective immunity in the natural host. In this study, one recombinant baculoviruses BacSC-E(rns) expressing histidine-tagged E(rns) with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64 was constructed and its immunizing efficacy was evaluated in a mouse model. After infection, E(rns) 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(rns) glycoprotein was successfully displayed on the baculoviral envelope. Vaccine tests in animals showed that BacSC-E(rns) elicited significantly higher E(rns) antibody titers in the immunized mouse models than the control group. This demonstrates that the BacSC-E(rns) vaccine can be used potentially against CSFV infections. This is the first report demonstrating the potential of E(rns)-pseudotyped baculovirus as a CSFV vaccine.

Seroprevalence of Toxoplasma gondii infection in dairy goats in Shaanxi Province, Northwestern China.

BACKGROUND: Toxoplasma gondii is an important zoonotic pathogen causing significant human and animal health problems. Infection in dairy goats not only results in significant reproductive losses, but also represents an important source of human infection due to consumption of infected meat and milk. In the present study we report for the first time seroprevalence of T. gondii infection in Guanzhong and Saanen dairy goats in Shaanxi province, Northwestern China. RESULTS: Sera from 751 dairy goats from 9 farms in 6 counties were examined for T. gondii antibodies with an indirect haemagglutination (IHA) test. Antibodies to T. gondii were detected in 106 (14.1%) serum samples, with antibody titres ranging from 1:64 to 1:1024. Seropositive goats were found in all 9 farms and seroprevalences in Guanzhong (16.3%, 75/461) and Saanen (10.7%, 31/290) dairy goats were not statistically significantly different. All the factors (sex, age and location) reported in the present study affected prevalence of infection, and seroprevalence increased with age, suggesting postnatal acquisition of T. gondii infection. CONCLUSIONS: The results of the present survey indicate that infection by T. gondii is widely prevalent in dairy goats in Shaanxi province, Northwestern China, and this has implications for prevention and control of toxoplasmosis in this province.

[Construction of an infectious clone of pseudorabies virus strain ZJ genome maintained as a bacterial artificial chromosome].

pHA2 plasmid sequence,with Bacterial Artificial Chromosome(BAC) vector and the GFP expression cassette, was introduced into the UL23(TK) gene of Pseudorabies virus(PRV)strain ZJ by homologous recombination,and the recombinant PRV (rPRV-HA2) was confirmed and isolated by plaque purification. The circular genome of rPRV-HA2 was electroporated into Escherichia coli strain DH10B and then the PRV BAC (pPRV) was recovered. The transfection of pPRV into VeroE6 cells resulted in productive infection. The rescued virus isolated following transfection was indistinguishable from rPRV-HA2 in cytopathic effects (CPE) and replication curve in vitro. The growth kinetics of the viruses indicated that partial deletion of TK gene and BAC vector insertion had no effect on the viral titre and plaque size in vitro. The PRV BAC system will enable quick and reliable manipulation of the viral genome for the functional investigation on the PRV genes and the development of PRV vector in vaccine.

[Construction of recombinant fowlpox virus expressing E0 gene of classical swine fever virus shimen strain and the animal immunity experiment].

The CSFV E0 gene was amplified from the plasmid pMD18-T-E0 by PCR and cloned into the FPV-P11 and FPV-pSY. The identified recombinant DNA was transfected into chicken embryo fibroblasts (CEF) to package Fowlpox virus. E0 gene was confirmed to be integrated into the genome of recombinant Fowlpox virus by PCR, and Western blot was employed for detection of E0 expression in the chicken embryo fibroblasts infected with recombinant Fowlpox virus . The results of ELISA showed that systemic immune response to CSFV could be induced effectively after the mice were immunized three times with recombinant Fowlpox virus through celiac route, the titer of antibody was 1 : 4096. The protection experiment showed that 75% of piglets immunized three times with recombinant Fowlpox virus were survived, indicating that the recombinant Fowlpox virus was effective. This paper lays foundation for the study of CSFV live vector vaccine.

[Construction and sequencing of full-length cDNA clone of swine vesicular disease virus strain HK'1/70].

By RACE, 2 overlapping cDNA fragments (3'PCR and 5'PCR fragments) covering the full genome of swine vesicular disease virus strain HK'1/70 were amplified from total RNA extracted from experimentally infected suckling mice. These fragments were cloned into pGEM-T Easy vector, respectively. 5'PCR fragment was digested by enzymes of Aat II and BssH II, and the Aat II-BssH II-digested 5'PCR fragment was obtained and cloned into the recombinant pGEM-T Easy vector containing 3'PCR fragment,the recombinant plasmid encoding full-length cDNA of SVDV HK'I/70 strain was then obtained and sequenced. The results showed that the complete genome of HK'1/70 was 7401 nucleotides (nts) long (excluding the poly (A) tract) which encodes a single polyprotein of 2185 amino acids, a 5'u ntranslating region (UTR) of 743 nts, a 3'UTR of 102 nts and a poly (A) tail at least 74 adenines. T' promoter was added at the 5'e nd of the full-length cDNA and an additional Pspl406I restriction site was added at the 3'e nd of poly (A) tail. The nucleotide and amino acid sequences were compared and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that HK'1 /70 belonged to the second antigenic group. SVD virus was antigenically closely related to Coxsackie B5 virus, and located on the branches of CB5 evolutionary tree. Successful construction of full-length cDNA clone of SVDV HK'1/70 strain lays foundation for rescuing SVDV effectively and enables further research of SVDV on molecular level.