Phylogenetic analysis of PRRSV
PRRSV was detected in 109 samples, which consisted of 47 samples for PRRSV-1 and 62 samples for PRRSV-2. Twenty samples were negative for PRRSV. Through ORF5 analysis, the PRRSV-1 samples belonged to pan-European subtype 1 and were classified into subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%) (Fig. 1). The PRRSV-1 nucleotide sequence homologies of ORF5 to Lelystad were 79.74–87.23% and 90.69–94.78% for subgroup A and subgroup C, respectively. The nucleotide sequence homologies of ORF5, ORF6, and ORF7 among the different PRRSV-1 samples were 79.59–99.83%, 85.85–100%, and 86.52–100%, respectively. The PRRSV-2 samples by ORF5 analysis were classified into lineage 1 (25/62, 40.32%), Kor C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%) (Fig. 2A). The PRRSV-2 nucleotide sequence homologies of ORF5 to VR2332 were 79.88–82.52%, 78.71–83.90%, 84.63–85.77%, 95.54–99.33%, and 82.78–86.14% for lineage 1, Kor C and B, lineage 5, and Kor A, respectively. The nucleotide sequence homologies of ORF5, ORF6, and ORF7 among different samples of PRRSV-2 were 74.81–100%, 83.21–99.81%, and 79.66–100%, respectively. Among lineage 1, 22 samples (22/62, 35.48%) could be classified into sublineage 1.8 (NADC30-like) and three samples (3/62, 4.84%) belonged to sublineage 1.6 (Fig. 2B). The mean evolutionary rates of 238 Korean PRRSV-1 and 319 PRRSV-2 were 6.931 × 10-3/site/year (95% HPD intervals from 5.8521 × 10-3 to 7.9748 × 10-3) and 5.131 × 10-3/site/year (95% HPD intervals from 4.516 × 10-3 to 5.7664 × 10-3), respectively.
Amino acid analysis of GP5 and M protein from PRRSV-1
Previous studies identified one neutralizing epitope at amino acid (aa) 29–35 in GP5, which was reported to be 29WSFADGN35 in the Lelystad strain. Additionally, there were T cell epitopes and four B cell epitopes (GP5-I, GP5-II, GP5-III, and M-I) in GP5 and M protein of PRRSV-1 (48-51). As shown in Fig. 3, the neutralizing epitope was conserved with a low level of entropy. Subgroup A did not show much variation in the neutralizing epitope region, while only the 20R44-37-1 sample belonging to subgroup C showed variation in position 31 (31F → 31S) and 35 (35N → 35S). By contrast, the GP5-III and M-I regions were variable with a high level of entropy, indicating genetic diversity (Fig. 3). A total of 16 codon sites were positively selected in the GP5 and M proteins of PRRSV-1 (Table 2). Interestingly, one positive selection site at position 35 was included in the neutralizing epitope region (positions 29–35), and two positive selection sites at positions 56 and 60 were included in the T cell epitope region (positions 53–75). Positive selection sites at positions 5, 7, 9, 20, 35, 36, and 104 in GP5 were observed with p-value < 0.01.
Amino acid analysis of GP5 and M protein from PRRSV-2
Both a decoy epitope and a neutralizing epitope located at GP5 of PRRSV-2 have been identified, comprising residues 27–30 (27VLVN30) and residues 37–45 (37SHLQLIYNL45), respectively (Ostrowski et al., 2002). The diversity of amino acid sequences occurred in two previously identified B cell epitopes, aa 1–15 and aa 187–200, and two previously identified T cell epitopes, aa 117–131 and aa 149–163 (de Lima et al., 2006; Vashisht et al., 2008; Zhou et al., 2009). As shown in Fig. 4A, critical amino acid variations in the B cell and T cell epitopes were also found in GP5 of PRRSV-2. In the decoy epitope compared with the VR2332 strain, significant diversity was found with higher amino acid entropy. Interestingly, a specific substitution at position 44 (44N → 44K) was found in the 21R2-15-1 sample belonging to lineage 1. In addition, 18R10-16-2 and 20D007-2 samples belonging to Kor C and 21D82-1 sample belonging to lineage 1 had a cysteine at position 102 in GP5. The 18R10-16-2 sample belonging to Kor C and 20D253-2 sample belonging to lineage 1 have an arginine at position 104 in GP5. Previous studies identified two B cell epitope regions at positions 10 and 70, and three T cell epitope regions at aa 9–23, aa 33–47, and aa 57–71 of M proteins (52-54). The M protein region was relatively more conserved compared with that of GP5 (Fig. 4B). A specific substitution at position 8 (8F → 8L) was found in the 19R44-3-2 sample belonging to lineage 5. In addition, amino acid mutations and higher amino acid entropy at position 10 in 50 PRRSV-2 and position 70 in 11 PRRSV-2 were found. Positive selection pressure analysis confirmed 18 codon sites in GP5 (Table 2). Four positive selection sites at positions 2, 5, 6, and 15 were included in the B cell epitope region (positions 1–15), two positive selection sites at positions 28 and 30 were included in the decoy epitope region (positions 27–30), and five positive selection sites at positions 32, 33, 34, 35, and 59 were included in hypervariable regions 1 and 2. Subsequently, two positive selection sites at positions 102 and 104 were included in the B cell epitope regions, and one positive selection site at position 151 was included in the T cell epitope region (positions 149–163). A total of four codon sites were found to be positively selected in M protein of PRRSV-2 (Table 2). One positive selection site at position 10 was included in the B cell epitope region, and three positive selection sites at positions 15, 16, and 66 were included in the T cell epitope regions (Table 2).
Sensitivities and specificities of three one-step real-time RT-PCR assays
The sensitivities of three one-step real-time RT-PCR assays with two reference strains (Lelystad and LMY strain) were estimated to be 100 TCID50/100μL. In the specificities of all one-step real-time RT-PCR assays determined by testing respiratory disease-causing viruses, such as PCV2, PCV3, CSFV, PPV, and SIV, no cross-reactivity was observed (data not shown).
Clinical evaluation of three one-step real-time RT-PCR assays
Twenty PRRSV-negative clinical samples showed the same results in all one-step real-time RT-PCR assays. As shown in Table 3, the A test detected 100% of PRRSV-1 (47/47) and PRRSV-2 (62/62) in the samples. The B test detected 100% of PRRSV-1 (47/47) and 98.39% of PRRSV-2 (61/62) in the samples. Only 18D283-1 sample, belonging to Kor A of PRRSV-2, was not detected in the B test. When identifying inhibitors in the 18D283-1 sample, none of the diluted samples was detected (data not shown). Meanwhile, the C test detected 72.34% of PRRSV-1 (34/47) and 69.35% of PRRSV-2 (43/62) in the samples. There were 13 undetected samples of PRRSV-1 belonging to subgroups A (n = 12) and subgroup C (n = 1), and 19 undetected samples of PRRSV-2 belonged to Kor C (n = 8), Kor A (n = 4), lineage 1 (n = 4), Kor B (n = 2), and lineage 5 (n = 1). On comparison with the nucleotide sequences of the primers and probe in the C test, several nucleotide mismatches were observed in the forward primer sequences. The forward primer against PRRSV-1 was located at positions 14,792–14,809 in ORF7 of the Lelystad strain. Several mismatches were observed of nucleotide positions 14,792 (G/A), 14,795 (C/T), 14,798 (C/T), 14,801 (C/T), 14,804 (C/T), 14,805 (C/T), 14,806 (A/T), and 14,808 (A/G). The forward primer against PRRSV-2 was located at positions 15,257–15,274 in 3′ end of ORF7 and 3′-untranslated regions (UTR) of the VR2332 strain. Several mismatches were identified at nucleotide positions 15,277 (A/G), 15,261(T/C), 15,263(G/A/T), 15,265(C/T/A), 15,267(G/A), 15,268(G/T/A), 15,269(C/T), 15,270(A/G) 15,271(T/C), 15,272(T/C), and 15,274(C/T). In particular, the 18R10-52-1 sample, belonging to Kor C of PRRSV-2, has 15 nucleotide deletions in the detection region of the forward primer.