Characterization of Two Immunodominant Antigenic Peptides in NSP2 of PRRSV-2 and Generation of a Marker PRRSV Strain Based on the Peptides

Figures

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  Figure 1. Systematic indel patterns of NSP2 based on 907 PRRSV-2 sequences. The indel patterns of NSP2 are divided into five main categories. The arrows and numbers indicate the indel locations in NSP2 using the position in the VR-2332 strain as a reference. Values on the right represent the numbers of strains.

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  Figure 2. Insertions and deletions in NSP2 of circulating PRRSV-2 or the MLV strain. VR-2332, the prototype of PRRSV-2, was used as a representative strain. Strains are listed on the left. The positions labeled in the figure are preferred to the corresponding position in VR-2332. Two regions (named m1B and m2B) at 562–627 and 749–813 are universal in PRRSV-2. On the left is the name of the representative strain of each indel type.

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  Figure 3. Antigenicity analysis and cross-reactivity assessment of m1B and m2B. (A) Indirect ELISA of m1B or m2B peptides. ELISA plates were coated with synthesized peptides, whereas pig serum positive for HP-PRRSV or NADC30-like PRRSV was used as the primary antibody. (B) ELISA plates were coated with synthesized peptides, whereas positive serum for PRRSV, CSFV, PEDV, PRV, PPV, and PCV2 was used as the primary antibody. The black dotted line represents the cutoff value of 1B2B-ELISA. HP-PRRSV, highly pathogenic PRRSV; CSFV, classical swine fever virus; PEDV, porcine epidemic diarrhea virus; PRV, pseudorabies virus; PPV, porcine parvovirus; and PCV2, porcine circovirus type 2.

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  Figure 4. Immunoreactivity of m1B and m2B truncated peptides. (A) Schematic diagram of overlapping m1B1–m1B7 short peptides used for epitope mapping. The green box represents peptides that reacted strongly with PRRSV-positive serum, whereas the gray box represents peptides that reacted only weakly. (B) P/N ratio for m1B1–m1B7 in indirect ELISA. (C) Schematic diagram of overlapping m2B1–m2B7 short peptides. (D) P/N ratio for m2B1–m2B7 in indirect ELISA.

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  Figure 5. Sequence analysis of m1B and m2B. Representative strains of current circulating PRRSVs or modified live vaccines (MLVs) are shown. The positions of indels in m1B and m2B are labeled below the sequences, whereas overlapping short peptides (m1B1–m1B7 and m2B1–m2B7) are labeled above the sequences. A blue line indicates reactivity between the short peptide and PRRSV-positive serum, whereas a gray line denotes no reactivity.

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  Figure 6. Characterization of PRRSV m1B and m2B deletion mutants. (A) CPE and bright fluorescence of deletion-marker strains and parental PRRSV-infected MARC-145 cells. a, rHuN4-F112-m1B; b, rHuN4-F112-m2B; c, rHuN4-F112-C5-m1B-m2B; d, rHuN4-F112; e, rHuN4-F112-C5; f, mock. Bar: 200 μm. (B) Growth curves of the parental virus and deletion mutants in Marc-145 cells. Values represent the average of three independent experiments. Error bars indicate standard deviations. Significant differences between the rHuN4-F112-m2B and parental virus are indicated (#P < 0.05, ##P < 0.01); significant differences between the rHuN4-F112-m1B and parental virus are indicated (*P < 0.05, **P < 0.01).