Lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (PToV-HE) protein
© Pignatelli et al.; licensee BioMed Central Ltd. 2013
Received: 21 May 2013
Accepted: 13 December 2013
Published: 23 December 2013
Hemagglutinin-esterases (HE) are viral envelope proteins present in some members from the toro-, corona- and orthomyxovirus families, all related with enteric and/or respiratory tract infections. HE proteins mediate reversible binding to sialic acid receptor determinants, very abundant glycan residues in the enteric and respiratory tracts. The role of the HE protein during the torovirus infection cycle remains unknown, although it is believed to be important in the natural infection process. The phylogenetic analysis of HE coding sequences from porcine torovirus (PToV) field strains revealed the existence of two distinct HE lineages. In a previous study, PToV virus strains with HE proteins from the two lineages were found coexisting in a pig herd, and they were even obtained from the same animal at two consecutive sampling time points. In this work, we report antigenic differences between the two HE lineages, and discuss the possible implications that the coexistence of viruses belonging to both lineages might have on the spread and sustainment of PToV infection in the farms.
Toroviruses (ToV) are enveloped, positive single-stranded RNA viruses of cattle, horses, pigs and humans . They have been associated with enteric infections and diarrhea, especially in young animals and children , and are considered a potential zoonotic threat . The Torovirinae subfamily of the Coronaviridae family (order Nidovirales) comprises four species: equine torovirus (EToV), bovine torovirus (BToV), porcine torovirus (PToV) and human torovirus (HToV). The few epidemiological studies performed in different countries indicate high prevalences of these viruses [4–6]. The torovirus’s genome size (~ 28 kb) and organization are very similar to those of other coronaviruses, with two huge overlapping open reading frames (ORF) shaping the 5′-end of the genome, where the replicase/transcriptase machinery is encoded, and a final third of the RNA molecule hosting the coding sequences for the four structural proteins (from 5′ to 3′): spike (S), hemagglutinin-esterase (HE), membrane (M), and nucleocapsid (N) . By analogy to other nidoviruses, the S protein is considered to be the putative receptor binding molecule. Copies of this molecule form the large spikes protruding from the viral particles as shown by electron microscopy [8, 9]. The HE protein forms homodimers which make up the smaller spikes [5, 10]. This HE protein is a class I membrane glycoprotein of about 65 kDa that belongs to the receptor destroying enzyme (RDE) protein family. As an RDE protein HE provides reversible binding to glycosylated surfaces  due to its ability to bind sialic acids and catalyze the disruption of that binding by means of its acetyl-esterase activity. The 3D structure and sialic acid preference of PToV- (strain Markelo) and BToV- (strain Breda) HE proteins have been solved . Two main domains have been defined in the HE monomer of both proteins: the enzymatic acetyl-esterase region (E) and the receptor binding (R) or lectin domain.
Despite the great amount of knowledge acquired on this protein family, it is not yet clear what is the exact role of HE during the torovirus infectious cycle. The EToV, the only cell culture adapted torovirus, as well as different BToV strains that could be isolated in cell culture, all lack a functional HE protein [13–16] due to deletions or mutations in the HE gene, acquired during their adaptation to in vitro growth conditions. Thus, HE expression seems to be detrimental for in vitro culture of these viruses, as it occurs with the coronavirus murine hepatitis virus (MHV) . However, the maintenance of the HE protein in a vast majority of new PToV and BToV field isolated strains indicates that it has to play an important function during in vivo infections. Several hypothetical functions have been postulated for the ToV-HE: (i) being a viral co-receptor, (ii) digestion of mucus layers to allow the virus to reach the target cells in the respiratory and/or enteric tracts, (iii) release of viral particles bound to decoy receptors, (iv) influencing host/cell tropism, or as recently proposed by de Groot’s group, (v) acting as a molecular timer in the virus pre-attachment step to the host-cell .
Two PToV-HE lineages have been identified, with representative strains being Markelo and P4 . They share an amino acid sequence homology of 80%. Recently, during a longitudinal survey of PToV in a Spanish pig herd, several PToV-HE isolates representative of both lineages were identified , and similar findings were described from a survey performed in Korea . In the first case, the two PToV-HE lineages were detected even within the same animal at two sequential sampling time points, indicating that both PToV strains carrying different HE proteins coexisted on the same farm infecting the same piglets, and suggesting that the immune response generated against one PToV strain did not protect the animals against the infection by the other strain. To further investigate this hypothesis PToV-HE proteins corresponding to each HE lineage were expressed, characterized, and used to track the anti-HE response in the animals from the farm where the PToV strains were obtained. The analysis of serum samples by hemagglutination inhibition assay and ELISA revealed antigenic differences between the two HE lineages.
Materials and methods
Cells, viruses and antisera
BSC40 (African green monkey kidney cells) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% heat-inactivated neonatal calf serum (NCS), non-essential amino acids (1%), gentamicin (50 μg/mL), penicillin (100 IU/mL), streptomycin (100 μg/mL) and fungizone (0.5 μg/mL).
Vaccinia virus (VACV), strain Western Reserve (WR) , and the recombinant VACV (rVV) derivatives were propagated and titrated in BSC40 cells.
To obtain a polyclonal antiserum against PToV-HE (αHE), a New Zealand rabbit was inoculated with two different peptides coupled to Keyhole limpet hemocyanin (KLH). Peptides comprising residues 51–67 (CTNPSTPNSLDIPQQLC) and 152–162 (LTPPENIPSHC) of PToV-HE-Bres [GenBank: FJ232070]  were chemically synthesized at the Proteomics facility of our institution after selection of target PToV-HE antigenic regions by bioinformatic analysis with Protean software (DNASTAR Inc. Madison, WI, USA).
Pig serum samples
The serum sample collection and their treatment were previously described . The samples corresponded to 12 animals from three different litters (A, B and C; four animals per litter) of the same farm, and were collected at 1-, 3-, 7-, 11-, and 15-weeks of age. Serum samples from the corresponding sow of each litter (n = 3) collected at the first day of sampling were also obtained.
Sequence analysis and modeling
Sequences were retrieved from the NCBI database. The sequence analysis was performed by comparing an equal number of sequences related to each PToV lineage, obtained from viruses identified in three different geographical regions: Italy, Spain and Korea. Sequences representing the Markelo lineage were from the PToV strains Markelo [GenBank: GU299776.1], P78 [GenBank: AJ575367.1], 52.7 [GenBank: GU299776.1], 14.7 [GenBank: GU299775.1], 07-56-14 [GenBank: FJ555594.1] and 07-56-23 [GenBank: FJ555596.1]. The HE sequences representing the P4 lineage were from PToV strains P4 [GenBank: AJ575364.1], BRES [GenBank: FJ232070.1], 52.11 [GenBank: GU299777.1], 12.11 [GenBank: GU299773.1], 07-56-11 [GenBank: FJ555593.1] and 07-56-22 [GenBank: FJ555595.1]. Protein sequences were aligned using a ClustalW algorithm (MegAling, Lasergene, DNAstar, Inc).
Alignment modeling was performed on the Markelo HE structural model [PDB: 3I1K] using Pymol v1.1 (DeLano Scientific LLC). The amino acid residues that are conserved in all sequences from one lineage but are different from those in the strains corresponding to the other lineage are depicted in blue. Other residues that are conserved in all sequences or are non-lineage specific are depicted in red.
Generation of rVV expressing PToV-HE genes
pGemT plasmids with PToV-HE coding sequences from isolates 52.7 and 52.11  were used to sub-clone the indicated PToV-HE sequences into the pJR101 VACV transfer vector  by asymmetric digestion with restriction endonucleases Bam HI and Nco I. The resulting plasmids, pJR-HE52.7 and pJR-HE52.11, carried the HE genes under the control of the VACV synthetic early/late promoter (pe/L). Insertion of the HE genes into the VACV hemagglutinin locus (HA) was achieved by homologous recombination between the transfer vectors and the virus genome following standard procedures . The resulting rVV-HE52.7 and rVV-HE52.11 viruses were selected and grown following standard procedures to yield viral stocks that were titrated and stored at −80 °C until use. A control rVV harboring a disrupted HA locus (rVV-HA-) was generated in parallel under the same procedures using the empty pJR101 vector.
rVV expressing soluble forms of HE52.7 and HE52.11 proteins fused to the c-myc tag were also generated to facilitate protein purification by affinity chromatography. For this purpose, the sequences coding for the fusion proteins were cloned into the pJR101 vector, and the corresponding rVV were obtained as described above.
Western blot analysis
BSC40 cells infected with the corresponding rVV at a multiplicity of infection (MOI) of 5 plaque-forming units per cell (PFU/cell) were collected at 24 hours post-infection (hpi) in Laemmli sample buffer. Expression of PToV-HE proteins in infected cells was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot with the rabbit polyclonal αHE serum. After incubation with a horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich, Saint-Louis, MO, USA), the reactive bands were detected by chemoluminescence using the commercial ECL reagent (GE Healthcare, Uppsala, Sweden).
Subconfluent BSC40 cell monolayers were grown in 12 mm-diameter coverslips and infected with rVV-HE52.7, rVV-HE52.11, or the control virus rVV-HA- at an MOI of 5 PFU/cell. At 7 hpi, cells were washed, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. After a blocking step with 20% fetal calf serum (FCS) in PBS, cells were incubated for 1 h with αHE polyclonal antiserum diluted (1:1000) in PBS containing 20% FCS, washed three times and stained with Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes™, Life Technologies, Carlsbad, CA, USA) at 1:500 dilution. DAPI reagent (4′, 6′-diamidino-2-phenylindole) (Molecular Probes™) was used to stain cell nuclei. After washing with PBS, coverslips were mounted on microscope slides using the ProLong® Gold anti-fade reagent from Molecular Probes™. Images were captured with a confocal Radiance 2100 system (Bio-Rad, München, Germany) and processed using ImageJ  and Adobe Photoshop CS4 (Adobe System Inc., San José, CA, USA).
Cell extracts for HE functional assays
Confluent BSC40 cell monolayers seeded in 150 mm plates were infected with rVV-HE52.7 and rVV-HE52.11 at a MOI of 5. At 24 hpi cells were rinsed with PBS and harvested. After a centrifugation step at 3000 × rpm for 10 min, cell pellets were resuspended in TNE buffer (1 mM Tris-Cl, 75 mM EDTA and 25 mM NaCl) and subjected to three successive freeze-thaw cycles and sonication. The soluble fractions were stored at −80 °C until needed. To obtain esterase inactivated PToV-HE cell extracts, rVV infected BSC40 monolayers were treated with 5 mM diisopropyl fluorophosphate (DFP; Sigma-Aldrich) for 30 min at room temperature before scraping the cells. Any remaining excess of DFP was removed after thoroughly washing the cells with ice-cold PBS.
Protein concentrations in the cell extracts were determined by Bradford reaction (Bio-Rad Protein Assay) using known amounts of purified bovine serum albumin (BSA) as standards.
Specific acetyl-esterase activity of both recombinant HE proteins (HE52.7 and HE52.11) was tested by an in situ staining assay as previously described . BSC40 cell monolayers were infected with the corresponding rVV-HE and with rVV-HA- as the negative control. At 48 hpi viral plaques expressing HE were visualized after incubation with α-naphtyl acetate-Fast Blue BB solution (ANAE assay kit, Sigma-Aldrich) according to the manufacturer’s instructions. Cell monolayers were then stained with crystal violet solution (0.5% in 20% methanol) to visualize all viral plaques.
Sialate-O-acetylesterase activity of rVV-infected BSC40 cell extracts in TNE solution was determined with the synthetic substrate p-nitrophenyl acetate (p NPA; Sigma-Aldrich) as previously described . Briefly, cell extracts were incubated with 3 mM p NPA at 37 °C, and the hydrolysis of p NPA was recorded by reading the absorbance at 405 nm at different times. The enzymatic activity per μg of cell lysate, after subtracting the background activity present in the control cell extract infected with rVV-HA-, was calculated as previously described .
Hemagglutination and hemagglutination inhibition assays
These assays were performed in U-shaped 96-well plates (Nunc, Roskilde, Denmark). Mouse (Mus musculus, strain Swiss) blood obtained from the retro-orbital cavity of anesthetized animals was collected in two volumes of sterile Alsever solution to prevent clotting. Animals were handled following the guidelines of the Animal Experimentation Committee of our institution, in strict accordance with the Spanish law (RD 1201–2005). The red blood cells (RBC) obtained were washed with Alsever solution, counted and diluted in PBS to obtain a 10% stock solution (10% solution corresponds to 8 · 108 RBCs per mL). Hemmagglutination assays (HA) were set-up using two-fold dilutions of extracts from DFP-treated and untreated rVV-infected cells. Fresh mouse RBC in PBS solution were added (50 μL) to the HE containing wells and incubated for two hours at 4 °C. Hemagglutination was scored and documented by photography, and plates were then placed at 37 °C and a second image was taken after 1 h incubation at this temperature. The hemagglutination units (HAU) of HE containing cell extracts were established as the reciprocal of the highest dilution causing hemagglutination.
For the hemagglutination inhibition assays (HI), HE cell extracts (4 HAU in 25 μL PBS) were incubated (1 h at 3 °C) with two-fold serial dilutions of kaolin treated pig sera  prior to adding 50 μL per well of a mouse erythrocyte suspension (final concentration of 0.5%). The HI titer was defined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination by the viral antigen. Serum samples with HI titers higher than 3 log2 were considered positive.
Purification of c-myc-tagged HE proteins
Extracts from BSC40 cells infected with rVV-HE52.7-myc or rVV-HE52.11-myc, were prepared as described above in TNE buffer containing 1% NP40. The soluble fractions were transferred to new tubes containing anti-c-myc coated beads previously equilibrated in TNE buffer (MBL, Naka-ku Nagoya, Japan). After 1 h incubation at 4 °C the mixtures of bead suspension and cell lysates were centrifuged in spin columns, washed thrice, and proteins were eluted with a c-myc tag peptide. The reagents and the protocol for the protein purification procedure were provided by the supplier. The purified proteins were analyzed by SDS-PAGE and Western blot with the αHE polyclonal serum and anti-c-myc monoclonal antibodies to confirm their identity (Clontech, Takara Bio Inc., Otsu, Shiga, Japan), and by Coomasie blue staining of the gel to determine their purity and concentration using different amounts of BSA as the reference.
Enzyme-linked immunosorbent assay (ELISA)
An indirect ELISA was set up to detect antibodies against PToV-HE proteins in pig serum samples. The optimal protein concentration was established by checkerboard titration with positive and negative pig control sera. Paired rows of a 96-well microtiter plate (Maxisorp, Nunc) were coated overnight (4 °C) with purified HE52.7-myc and HE52.11-myc proteins diluted at a 1.25 μg/mL concentration. Plates were thoroughly washed after each step by rinsing the plates thrice with PBS containing 0.05% Tween 20 (PBST). After coating, plates were blocked for 2 h at 37 °C with PBST-3% BSA. Then, 50 μL of each serum sample diluted 1:100 in PBST-1% BSA was added in paired wells and incubated 1 h at 37 °C. A commercial goat anti-pig IgG secondary antibody conjugated to horseradish peroxidase (Sigma-Aldrich) was used. The enzymatic reaction was developed using o-phenylenediamine dihydrochloride (OPD-FAST™, Sigma-Aldrich), and stopped after 10 min by adding 50 μL/well of 2 N sulphuric acid. Optical densities at 492 nm (O.D.492) were recorded with a multichannel spectrophotometer (Titertek Multiscan MCC/340). As negative control serum, a pool of sera from caesarian-derived, colostrum-deprived (CD/CD) pigs kept under germ free conditions (spf) was used, and the positive control serum was a commercial porcine serum (AbD Serotec, Kidlington, UK) previously determined to contain antibodies against PToV . The ELISA cut-off was established for each antigen as the mean of the O.D. of negative control serum plus three times the standard deviation (HE52.7-myc cut off = 0.08; and HE52.11-myc cut off = 0.10).
A previously described indirect ELISA was used to detect antibodies to the highly conserved PToV nucleocapsid protein .
Statistical analyses of HI and ELISA sera reactivities obtained from animals grouped by ages against both PToV-HE52.7 and PToV-HE52.11 were performed using two tailed t Student’s. Means ± standard error of the mean (SEM) for each group are shown.
Expression of PToV-HE proteins
PToV-HE functional characterization
The lectin activity of HE52.7 and HE52.11 proteins was tested by hemagglutination assay using mouse erythrocytes. The hemagglutination assay was set up with two fold serial dilutions of DFP treated (DFP+) and DFP untreated (DFP-) cell extracts, starting from 2 μg per well of total protein. The lysate from cells infected with rVV-HA- served as the negative control. At 4 °C, both DFP+ and DFP- rVV-HE cell extracts were able to induce hemagglutination of mouse RBC at a similar HA titer, whereas the HA- cell extract did not hemagglutinate the mouse RBC at any of the amounts tested (Figure 3C). When plates were placed at 37 °C, the hemagglutination net induced by DFP- cell extracts was disrupted due to their acetyl-esterase activity at that temperature, however hemagglutination was maintained in DFP-treated HE containing wells, indicating that the acetyl-esterase activity was completely inhibited (Figure 3D).
The esterase activity and receptor binding results show that both recombinant PToV-HE proteins were biologically active, indicating the acquisition of proper conformational folding.
Inhibition of PToV-HE-induced hemagglutination by pig serum samples
To compare the reactivity of each serum sample against both HE52.7 and HE52.11 proteins the HI titers obtained against the two HE proteins were plotted in Figure 4H. While few samples had the same HI titers with both proteins (serum samples found over the diagonal), most of them showed preferential reactivity with one versus the other HE lineage (serum samples found at both sides of the diagonal) and even some serum samples showed specific reactivity against only one of the HE proteins (7 serum samples were positive only for the HE52.7 and 5 were specific for the HE52.11). These data clearly indicate that HE lineage specific amino acid differences within the receptor domain were enough to determine that antibodies developed against one lineage do not interfere with the receptor recognition by the other HE lineage.
Analysis of the antibody response to HE in pigs by ELISA
To further investigate the reactivity of antibodies in piglets’ serum samples against HE52.7 and HE52.11 proteins and the dynamics of antibodies against each of them, an ELISA method using purified myc-tagged HE proteins (HE52.7-myc and HE52.11-myc) as antigens was used. The analysis by SDS-PAGE confirmed the purity of both preparations and their reactivities with both αHE polyclonal serum and anti-c-myc monoclonal antibodies were confirmed by Western blot (see Additional file 1).
Antibody response to the PToV-N protein
The reactivity of the piglet’s sera against the highly conserved N protein using a previously standardized ELISA  has already been analyzed . For comparison with the anti-HE immune response here we provide the results of the analysis of sera from the individual piglets over time (see Additional file 2). Although there are differences in the magnitude of the ELISA titers among the different piglets, what is clear from these results is that once maternally acquired antibodies had vanished, which for the N protein occurs around weaning time (week 3 of age), all animals developed their own anti-N antibodies that can readily be detected in most animals by week 7. These findings were in agreement with those obtained by HI and ELISA using the HE proteins as antigen, and indicate that all animals become infected by PToV soon after weaning.
Despite the great advances recently made on HE knowledge, which include the elucidation of both BToV-HE and PToV-HE tridimensional structures , the exact role of the HE protein in the viral life cycle and the potential relevance of the differences between HE lineages in the immunological response to the virus remain unclear. Without an in vitro culture system and given the great difficulties and costs of in vivo research, the use of heterologous expression systems represents a useful and relatively inexpensive approach to study the PToV-HE protein. Here we used the recombinant VACV methodology to express two full PToV-HE sequences, HE52.7 and HE52.11, and their two corresponding soluble fractions attached to a c-myc tag. Those HE coding sequences had been previously identified during a thorough longitudinal study of PToV in a Spanish farm , and each of them were found to belong to one of the two defined PToV-HE lineages . These HE sequences were obtained from the same animal in sequential collection points. This finding indicated that both lineages co-existed on the farm  although with apparently different prevalences according to the age of the host. This result could lead to new possibilities to approach the PToV-HE behavior in the virus’ natural environment.
Both recombinant proteins show similar features regarding their molecular weight, glycosylation degree, and subcellular localization as those reported previously for PToV and BToV [10, 17], indicating that the recombinant HE proteins follow a correct biosynthetic pathway. In addition, the heterologous expressed HE proteins were fully functional as receptor binding-receptor destroying enzymes since both PToV-HE proteins were able to hemagglutinate mouse erythrocytes, but also to hydrolyze the acetyl-ester linkage of glycan chains, as well as from acetylated synthetic compounds like p NPA and ANAE.
The analysis of the amino acid changes found between both PToV-HE lineages shows that there are amino acid residues that are conserved in a lineage specific manner even among strains identified in very distant geographic areas (different European countries and Korea). This analysis also indicates that potential antigenic differences would be mainly determined by residues located at the receptor binding domain, and exposed on the surface of the protein according to the proposed structural model . Hence, to elucidate if HE lineage specific changes could determine antigenic differences on the receptor binding domain, the HE 52.7 and 52.11 proteins were used as model proteins in HI assays with field serum samples from the same farm where both lineages were detected. Significantly, by this assay most serum samples show preferential reactivity to one of the HE proteins, or even specific reactivity against only one of them (see Figure 4H), indicating the existence of antigenic differences between the two HE lineages. Antigenic differences between HE proteins from the two known BToV lineages have also been described .
In addition, to study the antibody response against the whole protein by a different approach, soluble c-myc tagged HE52.7 and HE52.11 proteins were generated by rVV methodology to obtain highly purified coating antigens that were used in ELISA to test the same field serum samples. Using both approaches, a high prevalence of antibodies against PToV-HE was observed in both sows and piglets. These results were in agreement with those obtained using an ELISA assay with the very immunogenic and highly conserved N protein as antigen .
In the present study, similar anti-HE response profiles over time were observed by both HE-ELISA and the more restricted lectin-specific HI test. A general decrease of antibody levels was seen from the first weeks of age until piglets’ reached week 7, related to the extinction of maternally derived initial immunoglobulins. At 11-weeks of age, immune levels recovered due to the development of the pigs’ own response to infections, and they increased at least until week 15. At this last sampled point, a general increase of reactivity against HE52.11 (P4-like) was found in animal sera from the three litters, indicative of the prevalence of this lineage upon time. Overall, similar antibody patterns were observed by ELISA against the HE proteins and the N protein, although a delay in both the extinction of maternally derived antibodies and the development of self-acquired antibodies against the HE was observed in all animals analyzed (see Figure 5 and Additional file 2).
The rising of antibodies to PToV antigens after weaning can be explained as a consequence of the animal grouping in livestock facilities for fattening purposes that provides the conditions for piglet infection and/or re-infection and the mixture of PToV strains in the same animal population. Our results indicate that the immune response developed against one of the PToV lineages could not protect against the infection by other PToV isolates carrying an HE protein belonging to a different lineage. Hence, the specificity of the piglet’s current immune response, its own or maternal, towards one or the other HE lineage at a given time could determine the PToV strain that could infect or prevail in the animal. Our hypothesis is that this PToV lineage alternation could explain the sustainment of both strains on the farm. In fact, even though the P4-like (HE52.11) strain was not found by molecular detection in piglets at the earliest ages , the high reactivity observed by ELISA in sows and young piglets and, more remarkably, by the HI in sow A and in 1-, 3- and 7-week old piglets from litter A meant that such a strain was already circulating in the herd from the very beginning of the sampling. Particularly, in piglet 52 the increasing reactivity against both HE proteins from week 3 to week 15 indicates a temporal coexistence of both types of virus in the piglet at around weaning time. The lack of anti-HE maternal antibodies with an HI capacity against either HE protein might have facilitated the establishment of both PToV strains early on in the piglet’s life. Although we described that the two PToV-HE lineages were detected within the same animal at two sequential sampling time points, the lack of detection of PToV-HE52.11 at week 7 could have been due to the low abundance of this virus at the beginning of life, while at later times it became predominant, and therefore easier to detect. Although the shift from PToV-HE52.7 to PToV-HE52.11 in the analyzed animals (Markelo-like to P4-like strains) seems to derive from immune pressure on the latter, the contribution of a potential better viral fitness provided by the former on older pigs’ tissue environments cannot be discarded.
The tendency to recombine modules of their genomes observed in Nidovirales, together with the extensive mutation rates found, like in other RNA viruses, would facilitate the evasion of immune responses and the rapid adaptation to new hosts and the new hosts’ environments. From this study, where two PToV HE genotypes were found co-existing on the same farm, we can speculate that the specificity of the immune response towards one or the other HE lineage in piglets at a given time could determine the PToV strain that prevailed and spread. However, the potential additional contribution of the immune response to other viral antigens in virus selection also has to be considered. Future studies with higher numbers of animals from different farms will be required to further support the proposed hypothesis.
Though the PToV-HE protein in vivo function/s are still to be undoubtedly defined, its persistence in field strains, the tendency to undergo recombination events and the different antigenic characteristics of both HE lineages indicate that HE protein from torovirus plays an important role in virus–host interactions with implications in immune protection that could explain the broad spread of this virus in the pig population, causing chronic infections/re-infections of the animals.
We thank Joquim Segalés for providing porcine field serum samples. We also want to thank Susana Plazuelo for her excellent technical assistance in the analysis of sera by ELISA. This work was supported by grants AGL2010-15495 and CONSOLIDER-PORCIVIR CSD2006-00007 from the Spanish Ministry of Science and Innovation. Jaime Pignatelli and Julio Alonso-Padilla were both recipients of contracts financed with founding from the CONSOLIDER-PORCIVIR research project.
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