Whole genome sequence of bovine ephemeral fever virus Turkey isolates and development of DNA vaccine to the virus

Bovine ephemeral fever (BEF) is an arboviral disease characterized by a short-term clinical picture that can lead to significant loss in high-yielding cattle and water buffalo. In this dissertation study, briefly bovine ephemeral fever virus (BEFV) which is isolated from the BEF outbreak in 2012 in Turkey genetic characterization is aimed at the advanced level. Furthermore, it was aimed to generate a recombinant vector containing BEFV glycoprotein (G) and BEFV nucleoprotein (N) gene regions and to stimulate the humoral immune response against these proteins in BALB/c mice immunized with these vectors. BEFV was amplified by RT-PCR with partial gene regions of 600-800 bp including the complete genome for genetic characterization. The amplified RT-PCR products were sequenced using both directions with the forward and reverse primers in the ABI 310 Genetic Analysis® System. The Gene sequences were aligned using ClustalW program and presented to the gene bank. The phylogenetic analysis process was compared for similarity with the different isolates performed by the MEGA5 software program. The results show that phylogenetic relationships between BEFV isolates are closely related to the geographical location. There was also no genetic recombination between isolates. For recombinant vector design and immunization studies, BEFV G gene and BEFV N gene were transferred to the pcDNA4/HisMax© TOPO® vector and these recombinant vectors propagated in TOP10 competent cells. pcDNA4-G and pcDNA4-N recombinant plasmids were firstly transfected into Vero E6 cells and expressions were demonstrated at the protein level by Western blot and immunoperoxidase tests and demonstrated at the mRNA level by RT-PCR test. The suitable plasmids were treated with lipofectamine 2000 and intramuscularly administered to BALB/c mice at days 0, 14, 21. Antibody response in immunized mice was measured by plaque reduction neutralization test (PRNT) and ELISA. According to the BEFV G and N indirect ELISA, only two of the seven animals in these groups exceeded the cutoff value. When the mean OD values at 450 nm absorbance of the pcDNA4-N and the pcDNA4-G immunized mice group were compared with the plasmid control group, the difference became significant at 30 days (p<0.001), (p<0.05). According to PRNT50 results, 1:20 (p<0.05) antibody response was obtained at 30 days in pcDNA4-G (100 µg) immunized mice, whereas this ratio was 1:80 (p<0.001) in mice given BEFV (1000 PFU/0.5ml). As a result, recombinant proteins were successfully produced and stimulation of the humoral response was induced in experimental mice immunized with recombinant vectors.