A novel restriction fragment length polymorphism (RFLP) -based method for rapid genotyping of bovine coronaviruses

Outbreaks of diarrhea and respiratory symptoms caused by bovine coronavirus (BCoV) have been reported worldwide, leading to significant economic losses. It is important to genetically analyze the spike (S) gene of this virus as it is closely linked to its antigenicity and virulence, in order to quickly understand the molecular characteristics of circulating BCoVs. A previous study showed that BCoVs can be easily genotyped using reverse transcription (RT)-polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis using AvaII and EcoO65I enzymes targeted on the polymorphic region in the S1 subunit of the S gene. However, we found that a total number of 19 BCoV strains, including three BCoV strains isolated in this study and 16 Japanese BCoV strains available in GenBank, could not be genotyped by the original RT-PCR/RFLP analysis in silico. This study aimed to modify the original RT-PCR/RFLP assay by introducing additional restriction enzymes (AvaII, EcoO65I, AfaI, and Bsp1286I) to more accurately genotype recent Japanese BCoV strains without sequencing. As a result, the newly designed RT-PCR/RFLP analysis we established led to 98.3 % (291 of 296 BCoV strains used in this study) highly improved the accuracy of genotyping of BCoV. Therefore, this modified RT-PCR/RFLP method is useful for rapidly monitoring BCoVs, as it is a simple, easy, and inexpensive analysis that does not require sequencing.