Molecular characterization of a new PToV strain. Evolutionary implications

Abstract

Toroviruses are emergent viruses, belonging to the Nidovirales order, that remain mostly ignored, despite they are able to infect different species of domestic animals and humans, causing enteric diseases and diarrhea. Thus far, only five variants of porcine torovirus (PToV) have been identified. In this report we describe the identification and partial characterization of a new strain of porcine torovirus (PToV-BRES) that was detected by RT-PCR in a swine faecal specimen from a farm in Brescia (Italy). The complete genes coding for the nucleocapsid (N), hemagglutinin-esterase (HE) and membrane (M) proteins were amplified, and sequence analysis showed that PToV-BRES is a new PToV strain that, based on the HE gene sequence, is phylogenetically related to P4 strain, that was up to now the only member of a distinct PToV lineage. The nucleocapsid protein from PToV-BRES was expressed in insect cells as a his-tagged protein, purified by affinity chromatography and used to develop an ELISA method to detect antibodies against PToV. This assay was evaluated using a serum collection including 45 samples from three commercial farms from Spain. High antibody prevalence against PToV was observed in the three farms, both in adult animals and in piglets, which could suggest that PToV might be endemic in Spanish porcine population. The ELISA method developed in this work could be useful in future epidemiological surveys about toroviruses.

Fig. 1

Immunolabeled PToV particles visualized by negative staining. Toroviral particles present in swine faecal specimens were agglutinated by incubating the clarified faecal suspensions with convalescent serum from an infected pig (αBRES). The aggregated particles were loaded on a grid by ultracentrifugation in a Beckman Airfuge, and treated with 10 nm gold-conjugated protein A, and negative stained with 2% PTA. The electron micrographs correspond the 1318/02 porcine faecal sample.

Fig. 2

Detection of PToV and BToV in field faecal samples by RT-PCR. Porcine (A) and bovine (B) torovirus genomes were detected by RT-PCR using ToV-M5′-ToV-M3 primers (lanes 1 in panels A and B) from faecal samples 1318/02 and 1812/03 respectively, in which torovirus-like particles had been observed by electron microscopy. Primers 593–620 were used as positive control for specific amplification of PToV (lanes 2 in panels A and B). Distilled water negative controls were processed in parallel in each step (RT, lanes 3, and PCR, lanes 4, in panels A and B). (C) PCR products amplified form PToV-BRES cDNA corresponding to ORF-N (lane 1), ORF-M (lane 2), ORF-M 3′ end plus the full ORF-HE (lane 3), and ORF-HE (lane 4). Ladder DNA (pb) was run in parallel and sizes in bp are indicated.

Fig. 3

Phylogenetic analysis of ORF N (A), HE (B) and M (C) from PToV-BRES. Sequences from ORF N, HE and M were aligned with torovirus sequences available in GenBank. Comparisons were performed by neighbour-joining method using 1000 bootstrap. The numbers represent the distance to the nearest node and bar indicate the number of nucleotide substitutions (×100).

Fig. 4

Expression and purification of recombinant N protein from PToV-BRES. A recombinant baculovirus carrying PToV-BRES N gene (rBac-PToV-N) was used to infect HighFive cells. Infected cells were harvested at 24, 48 and 72 hpi (lanes 2, 3 and 4, respectively). Uninfected cells (lane 1) served as negative control. Protein expression was analyzed by SDS-PAGE and Western blot using (A) a commercial αHis antibody and (B) serum from a pig naturally infected with torovirus (αBRES). (C) PToV-N protein was purified from HighFive cells infected with rBac-PToV-N by affinity chromatography. The purification steps were analyzed by SDS-PAGE and Coomassie blue staining. The cell extract (lane 1) was prepared by lysis with NP40 and the insoluble fraction (lane 2) was treated with 6 M guanidine. After centrifugation the soluble fraction was incubated with Talon™ resin 1 h at 4 °C. After 3 washing steps, the bound protein (lane 3) was eluted from the resin by three elution steps (lanes 5, 6 and 7) with 1 M imidazol. A small amount of protein remained bound to the resin after elution (lane 4). Arrowhead indicates the position of the recombinant protein. The sizes of the molecular weight markers in kDa are indicated at the left or right margins.

Fig. 5

ELISA evaluation. 45 porcine serum samples collected from farms located in (A) Navarra (n = 10), (B) Galicia (n = 15) and (C) Aragon (n = 20), were analyzed by ELISA and Western blot using PToV N protein (400 ng) and sera diluted at 1:100. In addition, sera were analyzed by virus neutralization test at two-fold serial dilutions using BEV. Neutralization titers are expressed as the log ND₅₀. Western blot strips are shown in the upper part of the figure, and ELISA and neutralization values for each serum sample are represented on Y and X axes, respectively. IgG ELISA cut-off (O.D. 492nm = 0.270) (- - -) and virus neutralization cut-off (log ND₅₀ = 0.6) (|) are indicated. The arrows indicate serum samples with controversial ELISA and neutralization values from the farms in Navarra (A) and Galicia (B).