Bovine torovirus: sequencing of the structural genes and expression of the nucleocapsid protein of Breda virus

Abstract

Breda virus (BRV), a member of the genus torovirus, is an established etiological agent of diarrhea of cattle, which is found as two separate serotypes, BRV-1 and BRV-2. In this study, a 7.5 kb fragment of the BRV-1 genome that bracketed the genes for the structural proteins of BRV was amplified by long RT-PCR and the amplicon purified and sequenced directly. Sequence analysis revealed the presence of four open reading frames (ORF) corresponding to the peplomer (S), envelope (M), and nucleocapsid (N) genes, and an ORF for 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 of 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 coli expression system. The resulting protein was purified by SDS-PAGE and used to immunize guinea pigs. Hyperimmune serum was reactive with bovine torovirus (BTV) and human torovirus (HTV) antigens. By immunoelectron microscopy, it was shown to aggregate broken but not intact torovirus particles from BTV-positive fecal specimens. By immunoblot, the hyperimmune serum reacted specifically with the 20 kD N proteins of both BTV and HTV, as well as with the expressed N protein. BRV-1 and BRV-2 immune sera from gnotobiotic calves, but not human convalescent sera from HTV-infected patients, reacted with the expressed 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 specimens.

Fig. 1

Agarose/ethidium bromide gel electrophoresis of products amplified by long RT-PCR using RNA extracted from a bovine fecal specimen positive for Breda virus-1 (BRV-1) and primers flanking the torovirus nucleocapsid and peplomer genes. Lane marked N represents a negative control (ddH₂O). DNA mass ladder (M) and lambda HindIII (λ) were used as molecular size markers.

Fig. 2

Schematic representation of the BRV-1 genome showing the open-reading frames for the four viral structural genes, the peplomer (S), envelope (M), hemagglutinin-esterase (HE), and nucleocapsid (N). Numbers indicate the genome positions of the first and last nucleotides of each ORF sequenced starting from the 3′ end of the genome. The two ORFs of the polymerase gene (POL1a and POL1b) have not yet been sequenced.

Fig. 3

Nucleotide sequence of the BRV-1 nucleocapsid gene and the 3′ non-coding region of the BRV-1 genome aligned with that of BEV. Identical nucleotides are shown as dots. The predicted amino acid sequence of the BRV-1 N protein is also shown, and initiation (>>>) and termination (<<<) codons are underlined.

Fig. 4

Representative SDS-PAGE gels (left lane) and corresponding immunoblots of guinea pig anti-BRV-1 N protein preimmune (gpPI) and hyperimmune (gpHI) sera with (A) purified bovine torovirus-negative (BTV−) and positive (BTV+) fecal specimens, and (B) a purified human torovirus-positive fecal specimen. (C) SDS-PAGE gel of the expressed BRV-1 N protein from the insoluble E. coli fraction (left) and corresponding immunoblots of gpPI and gpHI sera, human acute (huA) and convalescent (huC) paired sera, and bovine anti-BRV-1 preimmune (bPI) and hyperimmune (bHI) sera.

Fig. 4

Representative SDS-PAGE gels (left lane) and corresponding immunoblots of guinea pig anti-BRV-1 N protein preimmune (gpPI) and hyperimmune (gpHI) sera with (A) purified bovine torovirus-negative (BTV−) and positive (BTV+) fecal specimens, and (B) a purified human torovirus-positive fecal specimen. (C) SDS-PAGE gel of the expressed BRV-1 N protein from the insoluble E. coli fraction (left) and corresponding immunoblots of gpPI and gpHI sera, human acute (huA) and convalescent (huC) paired sera, and bovine anti-BRV-1 preimmune (bPI) and hyperimmune (bHI) sera.

Fig. 5

Immunoelectron microscopy of a purified bovine torovirus-positive preparation with (a) guinea pig anti-BRV-1 N protein preimmune serum showing intact unaggregated toroviruses and (b) guinea pig anti-BRV-1 N protein hyperimmune serum showing aggregated partly disrupted particles. Bars=100 nm.

Fig. 6

Representative dot blot of five bovine torovirus-positive (BTV+), five human torovirus-positive (HTV+), five bovine virus-negative (NEG), and five bovine rotavirus-positive (ROTA) fecal specimens using guinea pig anti-BRV-1 N protein preimmune (gpPI) and hyperimmune (gpHI) sera and rabbit anti-guinea pig antiserum.