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A pseudorabies virus (PRV) variant with enhanced pathogenicity has emerged in many vaccinated swine herds in China since 2011. rPRVTJ-delgE/gI, a previously described gE/gI-deleted PRV based on the PRV variant TJ strain, has been shown to be avirulent to pigs yet virulent to sheep. To ensure desirable biosafety, we further deleted the thymidine kinase (TK) gene of rPRVTJ-delgE/gI to generate a gE/gI/TK-deleted mutant rPRVTJ-delgE/gI/TK, and evaluated its pathogenicity and immunogenicity in susceptible animals. Groups of mice (n=5), sheep (n=3), and pigs (n=4) were inoculated with different doses of rPRVTJ-delgE/gI/TK or rPRVTJ-delgE/gI, and clinical signs, viral shedding, pathological changes, and serum antibodies were examined following inoculation. The results showed that rPRVTJ-delgE/gI/TK displayed higher safety than rPRVTJ-delgE/gI for mice (10(3)-10(6) TCID50) and sheep (10(5) TCID50), and pigs inoculated with rPRVTJ-delgE/gI/TK (10(5) TCID50) induced PRV-specific antibodies and protection against lethal PRV challenge comparable to those immunized with rPRVTJ-delgE/gI. In conclusion, rPRVTJ-delgE/gI/TK has the potential to be developed as a vaccine for controlling the currently prevalent PR in China., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS). Pseudorabies (PR) is also an important infectious disease in swine and sometimes co-infect with PCV2. An attenuated pseudorabies virus (PRV) has been successfully used as a vector for live viral vaccines. In this study, a recombinant PRV expressing ORF1-ORF2 fusion protein of PCV2 was constructed and its immunogenicity was tested in mice and pigs. The ORF1 and partial ORF2 gene of PCV2 Yu-A strain were amplified by PCR and inserted into a transfer vector. The recombinant transfer plasmid was co-transfected with the EcoRI digested genome of vector virus (PRV TK-/gE-/LacZ+) into IBRS-2 cells. The recombinant pseudorabies virus PRV-PCV2 was purified by plaque purification and identified by PCR and Southern blotting. Expression of the ORF1-ORF2 fusion protein by the recombinant PRV-PCV2 virus was demonstrated by Western blotting analysis. The growth properties of the recombinant virus in cells were similar to that of the parent vector virus. In animal experiments, PRV-PCV2 elicited strong anti-PRV and anti-PCV2 antibodies in Balb/c mice as indicated by PRV-neutralizing assay, anti-PCV2 ELISA and PCV2 specific lymphocyte proliferation assay, respectively. And PRV-PCV2 immunization protected mice against a lethal challenge of a virulent PRV Ea strain. In pigs, PRV-PCV2 elicited significant immune response towards PRV and PCV2 as indicated by PRV-ELISA, PRV neutralizing assay and PCV2 specific lymphocyte proliferation assay, respectively. This is a first step toward the development of a potential candidate divalent vaccine against PRV and PCV2 infections.