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.

Seroprevalence of porcine torovirus (PToV) in Spanish farms

“…The high seroprevalence results obtained here confirm previously reported observations from other studies where fewer samples were analyzed [14,16,17]. Although a low seropositivity to PToV has also been reported, that conclusion could have been due to the detection method used, which relied on a BToV antigen based ELISA [19].…”

“…A total of 2664 samples collected from 100 swine farms distributed over the entire territory of Spain (Figure 1) were tested on the basis of a previously described ELISA [16] for the presence of anti-PToV IgG using the nucleocapsid protein as antigen. All of them were intensive breeding farms except 8 locations, where pigs were raised in outdoor production facilities.…”

“…This ELISA methodology had been previously shown to be sensitive and specific for anti-PToV pig IgG antibodies, and showed a very good correlation with the EToV heterologous neutralization assay [16,17]. Sampling was designed to establish the seroprevalence in the Spanish farmed swine population.…”

“…The presence of specific IgG antibodies against PToV in the sera was tested using an indirect ELISA previously developed in our laboratory, which is based on the use of the highly conserved viral N protein produced as a recombinant antigen [16,17]. The ELISA had been optimized using PToV positive sera from naturally infected pigs and negative sera form caesarean-derived, colostrum-deprived pigs kept under germ free conditions, and the assay cut-off had been established after five independent experiments performed in different days at an optical density value at 492 nm (O.D.…”

“…In the same study, over 80% seroprevalence against PToV was found in adult sows in The Netherlands using a heterologous neutralization assay against EToV. Very high seroprevalences against PToV have also been observed on samples from Switzerland using a similar neutralization assay [15], and in Spain by means of neutralization as well as ELISA [16,17]. However, in the three above cited studies, the numbers of farms surveyed and of serum samples analyzed were low.…”