Bovine torovirus: Sequencing of the structural genes and expression of thenucleocapsid protein of Breda virus

Breda virus (BRV), a member of the genus torovirus, is an established etiol ogical agent of diarrhea of cattle, which is found as two separate serotype s, BRV-1 and BRV-2. In this study, a 7.5 kb fragment of the BRV-1 genome th at bracketed the genes for the structural proteins of BRV was amplified by long RT-PCR and the amplicon purified and sequenced directly. Sequence anal ysis revealed the presence of four open reading frames (ORF) corresponding to the peplomer (S), envelope (M), and nucleocapsid (N) genes, and an ORF f or a novel 1.2 kb gene located between the M and N genes. This new gene was identical in nucleotide sequence to the hemagglutinin-esterase (HE) gene o f BRV-2. With the exception of this new ORF, BRV-1 manifests 80% nucleotide sequence identity with the torovirus prototype, Berne virus (BEV) in the 7 .5 kb region from the 3' end of the genome that contains the genes for the structural proteins. A 504 base segment containing the ORF for the BRV-1 N gene was amplified by RT-PCR, and cloned into an Escherichia coil expressio n system. The resulting protein was purified by SDS-PAGE and used to immuni ze guinea pigs. Hyperimmune serum was reactive with bovine torovirus (BTV) and human torovirus (HTV) antigens. By immunoelectron microscopy, it was sh own to aggregate broken but not intact torovirus particles from BTV-positiv e fecal specimens. By immunoblot, the hyperimmune serum reacted specificall y with the 20 kD N proteins of both BTV and HTV, as well as with the expres sed N protein. BRV-1 and BRV-2 immune sera from gnotobiotic calves, but not human convalescent sera from HTV-infected patients, reacted with the expre ssed N protein by immunoblot. These findings were applied to the design of a dot blot assay that could specifically detect BTV and HTV from fecal spec imens. (C) 1998 Elsevier Science B.V. All rights reserved.