Identification and characterization of a porcine torovirus

Abstract

A porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genus Torovirus (family Coronaviridae, order Nidovirales), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning.

FIG. 1

Serum neutralization titers against ETV in sera from four piglets 2 to 11 weeks old.

FIG. 2

Serum neutralization titers against ETV in sera from three different sows (sows A, B, and C; one serum sample each at day 0) and nine piglets (three piglets per sow). On day 0, the piglets were 1 to 3 days old; on day 21, they were weaned. Note that the scales on the ordinate are different for the three graphs.

FIG. 3

Morphology of PoTV and immunogold labeling (controls include TGEV and PEDV) with different sera. (a) PoTV particles in a fresh fecal sample from a piglet 1 week after weaning. (b to h) Immunogold staining of PoTV with anti-BoTV serum and protein A–5-nm colloidal gold conjugate (b); PoTV with A40, specific for TGEV (c); PoTV with anti-PEDV serum (d); PEDV with anti-PEDV serum (e); PEDV with anti-BoTV serum (f); TGEV with A40, specific for TGEV (g); and TGEV with anti-BoTV serum (h). The bar applies to all of the micrographs.

FIG. 4

Genomic organization of toroviruses and a schematic outline of the strategies used for amplification and sequencing of the 3′ genomic region. The upper panel shows a schematic representation of the torovirus genome, with the genes represented by boxes. The genes for the polymerase (Pol 1a, Pol 1b), the spike protein (S), the membrane protein (M), the hemagglutinin-esterase (HE), and the nucleocapsid protein (N) are indicated. The lower panel shows a schematic outline of the RT PCR assay targeted to the NTR and the N-protein gene. The positions and orientations of the oligonucleotides on the PoTV genome are shown, as are the lengths of the products of the PCRs. Primer 593 was used only in the sequence analysis.

FIG. 5

Amino acid sequence alignment of the N gene products of PoTV, BoTV, and ETV.