Study Of Infectious Porcine Reproductive And Respiratory Syndrome Virus Expressed By Baculovirus

Porcine reproductive and respiratory syndrome virus (PRRSV) is causative agent of porcine reproductive and respiratory syndrome, which is characterized by the abortion and infertility in infected sows, and the increased mortality of piglets from respiratory disease. PRRS has been epidemic in the global swine population, and then resulted in enormously economical cost for the swine industry. Today, PRRS is prevented by innoculated with the vaccine against PRRSV in the mainland China. However, the cells used to product the vaccine were limited by foreign patent. To be free from the patent, a new vaccine against PRRSV should be developed. Here, the baculovirus was used to develop a new vaccine.1. Construction of the recombinant baculovirus with PRRSVA recombinant baculovirus, AcAPRRS, was constructed by the genomic cDNA of PRRSV APRRS strain and bac-to-bac system. After 5 times passage of AcAPRRS on sf9 cells, the chimeric sequence in AcAPRRS between baculovirus genome and PRRSV cDNA was amplified, and then analysed. It was confirmed that AcAPRRS was of good stability. The growth curve resulted from AcAPRRS and AcLacZ, the vector baculovirus as controlled virus, on sf9 cells showed that AcAPRRS was of the similar dynamic of growth as AcLacZ. The transcription and expression of PRRSV genome on sf9 cell was detected by Northern blot and indirect immune fluroscence assay. There was low level of transcripted subgenome and N protein in sf9 innoculated with AcAPRRS. These results showed that a stable recombinant baculovirus was obtained.2. Characterization of PRRSV derived from AcAPRRSMarc-145 cells were innoculated with AcAPRRS. The PRRSV N protein was expressed in the cells detected by IFA. Three days post innoculation, Marc-145 cells had obviously cytopathic effect. The PRRSV RNA was detected in the supernatant and the second passage supernatant of Marc-145 cells. The end sequences of the offspring virus, vASM , derived from AcAPRRS were analysed by 5'- Race and 3'-Race. vASM was of the same end sequences as the isolates of PRRSV typeâ…¡. The plaque morphology and growth dynamic of vASM were almostly indistinguishable from those of APRRS in Marc-145 cells. These results showed that baculovirus could have no effect on the characterization of the offspring virus derived from itself and be ideal vector for delivery of heterologous gene.3. Study of transduction of AcAPRRS into cells nonsusceptive to PRRSVBHK-21 cells and vero cells were innoculated with AcAPRRS. The PRRS N protein was expressed in some cells detected by IFA. Marc-145 cells innoculated with the supernatant of BHK-21 or vero could have cytopathic effect post two days. The PRRSV RNA was detected in the supernatant of BHK-21 cell, vero cells and Marc-145 cells. The growth curves of PRRSV producted on BHK-21 cells and vero cells were determinded. The highest titres of PRRSV was up to 104.5 TCID50/ml. These results showed that a virus would be generated in its nonsusceptive cells into which viral genome were deliveried by recombinant baculovirus. Thus, a new pathway was opened up to develop vaccine against PRRSV.