Genetic Stability And Immunizing Dose Of Recombinan Fowl Poxvirus Expressing E0-E2 Gene Of Classical Swine Fever Virus

Classical swine fever(CSF) or hog cholera,caused by the classical swine fever virus (CSFV),is one of the most important viral diseases that cause serious economic loss to the swine industry worldwide.Vaccine immunization is an effective method to control epizootic CSF.This study was designed to research the genetic stability and immunizing dose of recombinant fowl poxvirus expressing E0-E2 gene of CSFV.1.The recombinant fowl poxvirus rFPV-E0-E2 was serially passaged for 20 passages in specific-pathogen-free chicken embryo fibroblast(CEF) culture.The passage 5,10,15 and 20 were chosen to identify the genetic stability of the recombinant fowl poxvirus rFPV-E0-E2 by blue plaque assay,PCR amplification,gene sequence analysis and indirect immunofluoresence assay.The blue plaque assay showed that the rFPVs were pure.And E0, E2 gene could be amplified from these recombinant fowl poxvirus.The sequence analysis shoude that there were only one point mutation on E0 gene(139A→G) and another on E2 gene(413A→G) in 20^th passages virus.The expression of E0 and E2 genes in CEF infected with the rFPV were also identified by indirect immunofluoresence assay.The result above showed that the rFPV was stable within 20^th passages and the CSFV E0,E2 gene expressed stably in recombinant fowl poxvirus.2.Three batches of recombinant fowl poxviruses rFPV-E0-E2 were collected on CEF cells.Purification,contect of virus and expression of specific protein were drtermined,after the sterility detection was carried out.Sterility experiment showed that the recombinant fowl poxviruses was not contaminated by bacteria or mould and the blue plaque assay showed that the rFPVs were pure.Then the titers of recombinant fowl poxvirus were also detected,so that the TCID₅₀ of these virus were determined as 10^-7.5/0.025 ml,10^-9/0.025ml and 10^-9/0.025 ml.The expression of E0-E2 gene in CEF infected with the rFPV were identified by indirect immunofluoresence assay.All the results suggested that these recombinant fowl poxviruses could be used as original species to produce vaccines.Besides,in order to study the immune dose of recombinant fowl poxvirus in pigs,one batch of recombinant fowl poxvirus vaccine was produced and the LD₅₀ of CSFV strain Shimen was also determined as 10^-2.8/2mL in 50 60-day-old piglets.3.Variable immune quantity with same challenge dosage was used to determine the optimum immunizing dose of recombinant fowl poxvirus.Twenty three pigs were separated into 6 groups at random and immunized with different doses and procedares.28 days after immunity,pigs which was immuned was muscular injected with 100LD₅₀ of CSFV Shimen strain.After 21 days' observation,protective efficency was evaluated.The result demenstrated that when injected with CSFV Shimen strain,pigs twice immuned with 1.04×10⁷pfu recombinant fowl poxvirus obtained protective rate as 50%,indicating that the median protective dose(PD₅₀) of this vaccine in pigs was 2×1.04×10⁷pfu recombinant fowl poxvirus.