Isolation of bovine coronavirus (BCov) in cell cultures

Abstract

One of the most common viruses in the world that causes disease in cattle is the bovine coronavirus (BCoV). This virus is the causative agent of respiratory and gastrointestinal infections in newborn calves, resulting in significant economic losses in both dairy and meat farming. Considering the complex epizootic situation with the coronaviruses in the world and partial antigenic affinity of BCoV with coronaviruses of other species of animals and humans, the isolation of new strains of coronaviruses, their identification and optimization of cultivation conditions becomes extremely important and relevant. The aim of our research was to determine the features of methods of isolation of bovine coronavirus and to select methods for its cultivation in cell culture in order to obtain the virus with the highest titers of infectious activity. Isolation of BCoV was performed in monolayers of MDBK and the primary-trypsinized calf kidney culture cells, using 20 samples collected from calves with clinical signs of respiratory or/and gastrointestinal disease. 16 samples were positive for BCoV by means of Real-Time PCR test. Up to fifth serial passage, only 4 of these isolates presented typical syncytial cytopathic effect. It has been experimentally established that the continious calf kidney cell culture line (MDBK) and the primary-trypsinized calf kidney culture (CK) are suitable for BCoV isolation and accumulation. The infectious titer of bovine coronavirus at the level of the fifth passage in the cultures of MDBK and CK cells reached 5.54 ± 0.16 lg TCD50/ml and 5.59 ± 0.14 lg TCD50/ml, respectively. However, due to the high cost of obtaining primary-trypsinized cell cultures, this isolation method may be unacceptable to most pharmaceutical companies and laboratories. Also after 5 serial passages, the viral material was again examined in Real-Time PCR to confirm the isolation of BCoV - the study of 4 samples with a characteristic syncytial CPE had a positive result in Real-Time PCR. However, of the Real-Time PCR-positive 12 samples, the virus could not be isolated in continuous cell cultures of MDBK and Vero, as well as in primary-trypsinized cattle lung and kidney cell cultures. This fact may indicate the presence of different strains of BCoV circulation in farms in our country. Further research is planned to be focused on optimizing the methods and modes of BCoV strains isolation, as well as to identify and study the cultural properties of new strains of BCoV circulating in Ukraine. We will also continue the study of the obtained viral isolate for the subsequent development of tools for the diagnosis and immunoprophylaxis of coronavirus infection in veterinary medicine.