Produção e caracterização de cepas recombinantes do herpesvírus bovino tipo 5 defectivas na enzima timidina quinase e glicoproteína E

Abstract

Bovine herpesvirus type 5 (BoHV-5) is an alphaherpesvirus associated with meningoencephalitis, an important disease of cattle in South America. Differential modified live vaccines that allow differentiation between vaccinated and naturally infected animals have been used in programs for control and eradication of porcine and bovine herpesvirus infections in several countries. Like in other alphaherpesviruses, the enzyme thymidine kinase (TK) and the glycoprotein E (gE) are related with pathogenicity and virulence of BoHV-5. Thus, the present study aimed to produce and characterize BoHV-5 viruses defective in TK and gE genes out of a Brazilian wild-type strain (SV507/99). In the first part of this study, several BoHV-5 clones resistant to brivudin (BVDU), a nucleoside analog which select TK-deficient virus, were selected. One such clone (BoHV-5/R-27) demonstrated to be genetically stable in vitro, and displayed the same kinetics of replication, plaque size and morphology in cell monolayers compared to the parental strain. Moreover, BoHV-5/R-27 was shown to be attenuated for rabbits since no clinical signs were observed after intranasal inoculation. These results demonstrate that BoHV-5 is sensitive to BVDU; BVDU-resistant mutants can be selected, and the BoHV-5/R-27 resistant virus retained its ability to replicate in tissue culture and was attenuated for rabbits. In the second part of the study, three recombinants BoHV-5 strains defective in gE, TK and both genes were constructed using the strain SV507/99 as a background and were characterized in vitro. Homologous recombination in cell culture was used to delete and substitute the entire or part of the coding regions of gE and TK for the green fluorescent protein (GFP) and betagalactosidase (β-gal) genes, respectively. The absence of gE and TK genes was confirmed by immunoblotting and PCR, respectively. In vitro characterization of the recombinant viruses (BoHV-5 gEΔ, BoHV-5 TKΔ and BoHV-5 gE/TKΔ) demonstrated that the deletions did not affect their kinetics of replication when compared to the parental strain. However, recombinants BoHV-5 gEΔ and BoHV-5 gE/TKΔ produced smaller plaques than BoHV-5 TKΔ and SV507/99. This study demonstrated the viability of the recombinant BoHV-5 viruses, which can be further used in pathogenesis studies and for vaccine development as well. The third part of this study evaluated the immunogenicity in cattle of two inactivated oil-adjuvanted vaccines containing SV507/99 or BoHV-5 gE/TKΔ infected cell cultures. For this, forty calves (20 animals/group) were vaccinated on days 0 and 22 post-vaccination (pv) and serum samples collected at different time points pv were tested for neutralizing antibodies against the homologous BoHV-5 strain and against several heterologous BoHV-5 and bovine herpesvírus type 1 (BoHV-1) isolates. All vaccinated animals seroconverted after the second vaccination (mean titers of 17.5 for the SV507/99 group and 24.1 for the BoHV-5 gE/TKΔ group), and the neutralizing antibody titers remained up to day 116 pv, showing a gradual reduction. Cross-serology with heterologous BoHV-5 and BoHV-1 isolates indicated that both vaccinated groups reacted similarly to the same virus, but with higher magnitude against BoHV-5 isolates. These results demonstrated that the inactivated vaccine using the BoHV-5 gE/TKΔ recombinant induced a satisfactory serological response. Based on the fact that gE can be used as a negative serological marker, this strains can be an alternative as a differential vaccine. In summary, the presented results describe the production and characterization of BoHV-5 strains defective for two genes associated with virulence. The recombinant strains can be used in pathogenesis studies and constitute potential candidate for differential vaccines.