Preparation And Immunogenicity Research Of VLPs Based On IBDV JL Strain

Infectious brusal disease (IBD) is an economically significant and acutecontagious immunosuppressive disease, caused by infectious brusal disease virus(IBDV). Bursa of young chicken was the major target of IBDV. With IBDVproliferating in lymphocytes, the bursa became degeneration and necrosis, causingimmunosuppression and a variety of diseases. Huge economic losses caused by IBDhave happened to our poultry industry. Recent studies showed that the virulence ofIBDV is continually enhanced and an increasing trend of occurrence was found in ourcountry. Worse still is the emergence of virulent strain (vvIBDV) and variant strains(vIBDV) and the antigen mutation, which made the traditional vaccines have nocontrol over their pandemic. Therefore, the development of more effective and safervaccines has become an urgent need.IBDV strain JL was isolated from a poultry farm suspected outbreak of IBD inJilin province and verified by agar gel diffusion precipitation (AGP), RT-PCR,research of biological characteristics and animal experiments. The typical clinicalsymptoms and pathological changes of IBD were observed in chickens infected withvvIBDV strain JL, of which the mortality reached up100%. VP2gene of strain JLwas blasted with those of different virulence and serotypes logged in GeneBank and ahigh homology between strain JL and the vvIBDV at home and abroad was found. Inaddition, the SspⅠ restriction sites,which represented an key feature of vvIBDV,were found in strain JL and those vvIBDV. therefore, the IBDV strain JL wasconsidered as vvIBDV.To prepare IBDV VLPs, the recombinant plasmid pFast-VP2was generated firstlyby inserting the VP2gene of strain JL into pFastBacHAT. Then, by blue-whitescreening after pFast-VP2was transformed into competent E.coli DH10Bac cells, therecombinant bacmid DNA rBacmid-VP2was obtained, which were subsequentlytransfected into insect cell Sf9to produce recombinant baculovirus vBac-VP2. DNA of sf9cells infected with vBac-VP2was extracted and PCR result showed a specificband with an expected size. The Sf9cells infected with vBac-VP2were stainedpositive against IBDV antibody using the indirect immunofluorescence assay (IFA).Western blot analysis displayed a specific protein band of approximate51kDa, and theVLPs was observed by electron microscopy. The above results indicated that the VP2gene of IBDV was expressed in sf9cells and self-assembled into virus-like particles.This study paved the way for developing the IBDV virus-like particle vaccine.VLPs, VLPs+Poly IC were used for immunizing chickens at the age of14d as thesame as the B87live vaccine. The peripherals were blooded before primaryimmunization and weekly after that via jugular vein and were used for indirect ELISAantibody detection, neutralization test and lymphocyte proliferation test. The resultsshowed that the specific antibodies to IBDV were detected in the serum samples ofchickens inoculated with VLPs, VLPs+Poly IC and B87at7d post primaryimmunization and the levels increased significantly after booster immunization (P<0.05). High titer of neutralizing antibodies were also detected at14days afterprimary immunization in the three groups and kept a fast growing trend. Theirlymphocyte proliferation dynamics were also higher than those vaccinated with PBSor empty bacmid protein (P <0.01) and increased significantly after boosterimmunization (P <0.05). After challenge, chickens inoculated with PBS or emptybacmid protein exhibited typical clinical symptoms and pathological changes of IBDand the mortality rate was100%. While88.7%、80%、88.7%of chickens inoculatedwith VLPs, VLPs+Poly IC and B87were protected from vvIBDV, respectively.There was no significant difference in organ index among chickens inoculated withVLPs, VLPs+Poly IC, B87and those in blank (P>0.05). This study showed thegood prospects of VLPs in developing new and efficient IBD vaccines and providedreferences for the prevention and control of IBD.