The construction and characterization of bovine herpesvirus 1 expressing cytokines
This thesis investigated the potential of using Bovine Herpesvirus-1 (BHV-1) virus as a viral vector to express cytokines, and analysed the effects of these cytokines on BHV-1 infection and host immunity. Two constructs were generated by homologous recombination by using transfer vectors containing either EL-1β or bovine IFN-ɣ flanked by the 5' and 3' ends of glycoprotein C gene. Recombination occurred within the gC locus which created a recombinant virus with a gC minus (gC -) background phenotype that expressed the cytokine using the gC promoter. Molecular characterization showed that the recombinant cytokine genes were expressed with similar kinetics to a late BHV-1 protein; mRNA expression was detected at 5 hour post-infection followed by the detection of biologically active protein. These recombinant proteins had biological activity comparable to a recombinant protein standard. Both recombinant viruses, BHV1/1L1β and BHV-1/IFNɣ, exhibited 'in vitro' growth characteristics similar to a gC-minus virus. This indicated that the expression, of these cytokine did not affect BHV-1 growth. To analysis the 'in vivo' effects of these recombinant viruses, two experimental models were used. First, cattle were used to analyse the immune responses and viral pathogenicity during infection by recombinant BHV-1/IFNɣ. Similar levels of virus shedding and similar clinical responses were observed for both recombinant BHV-1/IFNɣ and gC-/LacZ+ viruses. BHV-1 was shown to be a potent inducer of boIFN-ɣ; in the nasal cavity and IFN-ɣ secretion correlated with the onset and duration of viral shedding. Analysis of cellular and humoral responses did not reveal any significant immune modulation during infection by BHV-1/IFN-ɣ. Serum IgG, mucosal IgA and antibody viruneutralization titers were similar between BHV-1/IFNɣ and control gC-/LacZ+ virus. In addition, gD specific proliferative responses and IFN-ɣ; ELISPOTs did not reveal any differences. After re-activation from latency with dexamethasone, viral shedding was similar for both BHV-1/IFNɣ and gC-/LacZ+ viruses. These results suggested that during a respiratory infection, the production of exogenous IFN-ɣ did not provide an advantage to the host. Also, BHV-1/IFN-ɣ was a stable vector during latency and recrudescence and expressed biologically active IFN-ɣ protein throughout the experimental period. A sheep model for BHV-1 infection was also used to evaluate the recombinant BHV-1 vector. A preliminary experiment showed that both wild-type BHV-1 Cooper virus and gC-/LacZ+ infected sheep, caused mild clinical signs and generated BHV-1 specific immune responses. Recombinant BHV-1/IL-1β and BHV-1/IFNɣ were then used to infect sheep. In this experiment, there was no difference in viral pathogenesis or the immune responses to the recombinant viruses and gC-/LacZ+ virus. Results in both sheep and cattle indicated that using BHV-1 as a viral vector to express bovine IFN-ɣ or IL-1β did not alter pathogenesis or the host immune response.
Doctor of Philosophy (Ph.D.)