Isolation in HRT-18 cells and molecular analysis of a BCoV strain from diarrheic feces of naturally infected calves

Abstract

Bovine coronavirus (BCoV) may cause acute diarrhea in newborn calves, leading to significant economic losses for cattle farmers. There are several diagnostic techniques used to detect BCoV in calf fecal samples, but virus isolation still has advantages for antigenic and genomic characterization. This study describes the isolation in HRT-18 cells and molecular characterization of Brazilian BCoV wild-type strains. Three fecal samples from diarrheic 30 day-old calves were inoculated in HRT-18 cell monolayers, which were then evaluated for HA titers and tested using seminested PCR followed by RFLP and sequencing. Two samples were successfully isolated and presented HA titers of 16 and 32 units per 25 μL. The results were confirmed using semi-nested PCR and RFLP. Molecular analyses identified a cell culture-adapted strain and a wild-type strain that were genetically similar (99%) to each other, but more distinct than BCoV strains circulating in other countries, even in the conserved N gene.