| Infectious bovine rhinotracheitis virus (IBRV), also known as bovine herpesvirus-1 (BHV-1), causes infectious bovine rhinotracheitis (IBR). This disease can lead to upper respiratory tract disorders, vaginitis, balanitis, abortion, conjunctivitis, encephalitis and immune suppressive. BHV-1 is the main causative agent of bovine respiratory disease complex (BRDC) that is also known as shipping fever. In addition, BHV-1 can induce immunosuppression in infected cattle that is prone to the animals for secondary bacterial infection like Pasteurella haemolytica, Haemophilus somnus and Pasteurella multocida. IBR causes significant decrease in the production performance of the infected cattle that leads to heavy economic losses to the cattle industry. Vaccination becomes one of the most effective tools for control of the infectious diseases. The ideal genetically engineered gene-deleted vaccines are characterized by that the virus was attenuated significantly, does not inhibit immune responses and maintains strong immunogenicity. Our previous work confirmed that IBR has a high prevalence level in our country cattle herd. To prevent the outbreak of this disease, we have successfully constructed the BHV-1 double gene-deleted vaccine. In this study, we hope to enhance the immunogenicity of this recombinant virus through the gene adjuvant.Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, which is generated by activated T lymphocytes, B lymphocytes, fibroblasts, macrophages, vascular endothelial cells and other cells. In the immune response, it can enhance antigen-presenting ability mainly through the increase of proliferation and differentiation of antigen presenting cells in particular dendritic cells.In our study, we introduce bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene into BHV-1 gG-/TK-/EGFP+ through homologous recombination to increase the the immunogenicity and protective efficacy of BHV-1 gG-/TK-/GM-CSF+. The main contents are described as follows:1. Construction of the transfer plasmid with foreign gene GM-CSF and upstream and downstream homologous gene arms of tk geneBovine GM-CSF was cloned into pcDNA3.1 vector and then we obtain the GM-CSF gene expression cassette containing the CMV promoter and BGH poly (A) terminator. Next we obtained the transfer plasmid by cloning the expression cassette with upstream and downstream homology arms of the tk gene into pcDNA3.1 vector. The transfer plasmid was identified successfully through restriction enzyme digestion and sequencing.2. Construction of the recombinant virus BHV-1 gG-/TK-/GM-CSF+By using the calcium phosphate transfection method we co-transfected MDBK cells with the viral genomic DNA of BHV-1 gG-/TK-/EGFP+ and linearized transfer plasmid bearing GM-CSF gene digested by Hind Ⅲ. Then we screened for the virus which had CPE but without green fluorescence, and the resultant virus was designated as BHV-1 gG-/TK-/GM-CSF+3. Identification of the recombinant virus BHV-1 gG-/TK-/GM-CSF+We identified successfully the correct insertion of GM-CSF gene expression cassette into the recombinant virus tk gene by using PCR and sequencing, transcription with reverse transcription PCR. But we didn’t detect expression of GM-CSF protein by Western blot, probably because the GM-CSF protein was secreted into the media (the integrated GM-CSF gene has the signal sequence) or expressed at a low level.4. Identification of the biological characteristics of BHV-1 gG-/TK-/GM-CSF+There was no significant change in plaque size between the parental strain BHV-1 gG-/TK-/EGFP+ and recombinant strain BHV-1 gG-/TK-/GM-CSF+. The viral titers of BHV-1 gG-/TK-/EGFP+ and BHV-1 gG-/TK-/GM-CSF+ in MDBK cells were 3.2×107PFU/mL and 3.5×107PFU/mL respectively. Both have similar growth curves. BHV-1 gG-/TK-/GM-CSF+ was continuously passaged for 20 generations in vitro and GM-CSF gene was detected by PCR every 5 generations and this gene maintained in all the passages. The results showed the GM-CSF gene remains stable in the recombinant virus.5. Study on the immunology of BHV-1 gG-/TK-/GM-CSF+ in rabbitsThe rabbits were inoculated with the parent strain BHV-1 gG-/TK-/EGFP+, the recombinant strain BHV-1 gG-/TK-/GM-CSF+and BHV-1 gG-/TK-/GM-CSF+with Seppic adjuvant respectively. Then we detected neutralizing antibody, ELISA antibody and the production of IL-4 and IFN-β. Results showed that both types of antibody could be detected at 7 days after inoculation and reached the highest levels in four weeks, but there is no significant difference among all groups. The serum IFN-β and IL-4 levels were checked 3,5 and 7 days after inoculation. The serum IFN-β in recombinant virus group was significantly higher than that in parent virus group at 3 and 5 days after inoculation (P＜0.05). There is no significant difference in IL-4 level among all groups. The recombinant virus with the Seppic adjuvant had a better immune effect compared with the recombinant virus without the adjuvant.After 28 days of inoculation, the rabbits were challenged with wt BHV-1 at a dose of 4×107 PFU. We observed the clinical signs such as general status, temperature change and virus shedding through nostrils. The results showed that the three groups of vaccinated rabbits can produce protection against the challenge. |
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