Lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (PToV-HE) protein

Abstract

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.

Figure 1

Sequence analysis of HE proteins from PToV strains representative of both lineages. (A) Multiple alignments of HE sequences. (B) Modeling over the Markelo HE structure (in red) of amino acid residues differing between Markelo and P4 lineages (in blue).

Figure 2

Expression of PToV-HE52.7 and PToV-HE52.11 proteins. (A) Western blot detection of HE proteins in extracts from BSC40 cells infected (MOI 5) with rVV-HE52.7, rVV-HE52.11 or rVV-HA-. Infected cells were fractionated by 10% SDS-PAGE, transferred to a nitrocellulose membrane and reacted with the αHE antibody. α-Tubulin signalling was taken along as a loading standard. Molecular size markers are given in kDa. (B) Immunofluorescence microscopy of BSC40 cells infected with rVV (MOI 5). Cells were fixed at 7 hpi, permeabilized and incubated with αHE antibody followed by a secondary antibody coupled to Alexa Fluor 594 (red). Cell nuclei were stained using DAPI (blue). Scale bar: 10 μm.

Figure 3

Functional characterization of recombinant PToV-HE52.7 and PToV-HE52.11 proteins. (A) ANAE in situ staining of rVV plaques produced in BSC40 infected with rVV-HE52.7, rVV-HE52.11 or rVV-HA- (~ 100 PFU per well). After the ANAE assay cell monolayers were stained with crystal violet to visualize all viral plaques. (B) Acetylesterase activity in extracts from BSC40 cells infected with rVV expressing PToV-HEs. Cells infected (MOI 5) with rVV-HE52.7 and rVV-HE52.11 or with the control virus rVV-HA- were harvested at 24 hpi, resuspended in TNE buffer and tested for acetyl esterase activity by incubation with 1 mM pNPA reagent. Background corrected hydrolysis of pNPA (in nmol), measured at 405 nm along a time span (min) is shown in the graph. (C-D) Hemagglutination assays with mouse RBC (0.5%) and 2-fold dilutions of both DFP-treated (+DFP) and untreated (−DFP) rVV-infected cell extracts resuspended in TNE buffer, recorded after incubation at 4 °C (C) and at 37 °C, (D). The negative controls (C-) correspond to wells where RBC were incubated with TNE buffer.

Figure 4

Analysis by HI assay of the reactivity of sera obtained from pigs at different ages against PToV-HE52.7 and PToV-HE52.11. Extracts from BSC40 cells infected (MOI 5) for 24 h with rVV-HE52.7 (panels A, C and E) or rVV-HE52.11 (panels B, D and F) and treated with DFP were diluted to contain 4 HAU in 25 μL PBS and incubated (1 h at 37 °C) with two-fold serial dilutions of pig sera prior to adding the mouse RBC. Serum samples were obtained from animals belonging to three litters (litter A, panels A and B; litter B, panels C and D; litter C; panels E and F) at different ages (1, 3, 7, 11 and 15 weeks) and from their sow (one-week post-farrowing). (G) Percentage of HI positive animals against PToV-HE52.7 (blacK bars) and PToV-HE52.11 (grey bars) over the period studied. (H) Comparative analysis of HI titers of each serum sample against PToV-HE52.7 and PToV-HE52.11 proteins. The results shown in the graphs are representative of two independent experiments.

Figure 5

Reactivity of sera obtained from pigs at different ages against PToV-HE52.7 and PToV-HE52.11 determined by ELISA. Purified HE52.7-myc- and HE52.11-myc proteins were used as coating antigen (62.5 ng/well). The same serum samples analyzed in Figure 4 were used in the ELISA at a 1:100 dilution. IgG ELISA reactivities against HE52.7-myc- (panels A, C and E) and HE52.11-myc (panels B, D and F) of serum samples from pigs and their sows are represented.

Figure 6

Paired comparisons of the results of pig sera reactivities against PToV-HE52.7 and PToV-HE52.11 determined by HI and ELISA. Graphic representation of the mean HI (A) and ELISA (B) titers of sera grouped by pig ages against both PToV-HE52.7 (black bars) and PToV-HE52.11 (grey bars). Means ± standard error for each sample are shown. Asterisks indicate statistically significant differences of sera reactivity against the two proteins (**p < 0.05, *p < 0.1; Student’s t test). The dotted lines indicate the respective cut off values determined for each assay.