Detection, isolation, molecular characterisation and immune response to influenza D virus in cattle

Influenza D virus (IDV) causes respiratory system infections in cattle. IDV has been reported globally in several countries including Turkiye with various degrees of morbidity. It has been reported that IDV contributes to Bovine Respiratory Disease Complex (BRDC) in cattle which causes great economical loses. There are no vaccines available for IDV yet and hereby strategies for prevention and control of IDV include hygiene and good management pratices. In this thesis, detection, isolation, molecular characterisation and immun response to IDV in cattle were aimed. For this purpose, samples (n=279) was collected from Marmara region of Turkiye. These samples were collected from animals with respiratory disease symptoms and include nasal swabs and blood. Virus isolation and antibody response were investigated using molecular and serological techniques. Followed by IDV-RNA detection by real time RT-PCR, virus isolation on cell culture and embryonated eggs were performed. After virus isolation, positive samples were confirmed by RT-PCR and phylogenetic anaylses were carried out on partial and whole HEF gene. In addition, hemaglutination (HA) titers were determined and viral proteins were separated by SDS-PAGE. For antibody response, ELISA and hemagglutination inhibition (HI) tests were performed. A serum with high antibody titre was used in Western blotting to determine the molecular weight (kDa) of which viral proteins binds to antibodies produced by IDV post- infection. According to the results, out of 279 nasal swabs, IDV-RNA was detected in five cattle (1,79%). These 5 samples found to be positive by real time RT-PCR were subjected to virus isolation on HRT-18 cell cultures and embryonated eggs. Sequences of partial HEF gene was obtained from 5 samples and whole HEF gene of 4 samples. Sequences reported from different regions and countries in the world were provided from GenBank and these sequences were used for the phlogenetic analyses to generate phlogenetic trees with the sequences obtained in this thesis. Positive samples which were collected in 2020-2021 were aligned in D/OK lineage and samples collected in 2024 were aligned in D/Yama2019 lineage. ELISA results indicated that IDV antibodies were detected in 79 (28,3%) of 279 cattle sera. Positive samples were also analysed by HI to determine hemagglutination inhibition titers and HI unites were found to be between 128 and 512. A serum with high HI titer was used for western blotting to investigate antibodies bind to which viral proteins and antibodies were reacted with a 100kDa protein of IDV. In conclusion, IDV which has been circulating in Marmara region was isolated and the seroprevalance was investigated by ELISA, HI and Western blotting for the first time and antibodies to IDV were detected in 28,3% cattle. In Western immunoblotting studies, it was observed that antibodies formed as a result of field infection reacted with the 100 kDa protein of IDV, which indicate that this protein should be taken into consideration in vaccine studies. In addition, as a result of whole genome studies of the HEF gene, it was determined that the virus was similar to the D/OK and D/Yama2019 strains. Therefore, it is important to use these viruses in terms of vaccination strategies in case of production of autogenous vaccines specific to our country.