Comparative analysis of the nsp2 and ORF5 genes
To evaluate the similarity of the deletion site in the nsp2 gene between EDRD1 and HP-PRRSV (strain 10186-614), we conducted comparisons of these with two other reference strains, namely, VR-2332 and JXA1. An amino acid alignment of the partial sequences of nsp2 in these four strains, in order of their pairwise identity, is shown in Figure 1. Strain 10186-614 contained two discontinuous deletions in the translated protein with deletion sizes of 1 and 29 amino acids corresponding with amino acid positions at 482 and 538–567, respectively, in VR-2332 nsp2. By contrast, EDRD1 contained one continuous deletion of 39 amino acids corresponding with amino acid positions 509–547 in VR-2332 nsp2. Two potential N-glycosylation sites were detected in VR-2332 and EDRD1 at amino acid positions 325 and 697 of VR-2332 nsp2. However, amino acid position 325 is a commonly mutated asparagine-to-lysine residue in JXA1 and 10186-614 strains. The conserved cysteine residue proposed to interact with the matrix protein and the protease sequence that is conserved within PRRSV and equine arteritis virus were well conserved among all four strains.
ORF5 encodes glycoprotein (GP) 5, which is frequently used for the diagnostic identification of PRRSV . To elucidate the genetic distances between 10186-614 and EDRD1, we aligned GP5 among the four strains (Table 1). Alignment of PRRSV GP5 determined shared amino acid identities ranging from 87.9% to 89.5% between VR-2332 and other strains and from 88.9% to 89.9% between EDRD1 and other strains. Additionally, strains 10186-614 and JXA1 shared 97.0% identity.
Clinical scores in the EDRD1-inoculated group (Group 2) were significantly lower than those in the group without EDRD1 inoculation (Group 3) from 3 days post-infection (dpi) till the end of the experiment with strain 10186-614 (Fig. 2A). After 10186-614 challenge, pigs in Group 3 exhibited periocular edema and redness of the skin on the trunk. These pigs demonstrated a poor appetite and respiratory signs and became seriously ill with coughing and abdominal breathing. These clinical signs persisted through the end of the experiment. A proportion of the animals in Group 3 lay collapsed in the recumbent position and exhibited intermittent thrill. One of these (pig G17) was euthanized on 12 dpi due to difficulty drinking water by itself. By contrast, three of five pigs in Group 2 transiently showed loss of appetite and tachypnea between 3 and 12 dpi, but the pigs recovered by 13 dpi. No clinical signs were observed in animals not challenged with strain 10186-614 (Groups 1 and 4).
Daily weight gain and rectal temperature
The mean daily weight gain of the pigs in Group 3 was significantly lower than that of Group 2 between 9 and 13 dpi (Fig. 2B). By the day of necropsy (16 dpi), the mean weight gain was 9.00 kg for Group 1 (EDRD1 inoculation without HP-PRRSV challenge), 10.00 kg for Group 2 (EDRD1 inoculation and HP-PRRSV challenge), 5.13 kg for Group 3 (HP-PRRSV challenge without EDRD1 inoculation), and 9.60 kg for Group 4 (uninfected controls).
The mean rectal temperature in two of five pigs (pig G9 and G12) in Group 2 increased transiently at 3 dpi, whereas that of all animals of Group 3 rapidly increased until plateauing 3 dpi (Fig. 2C). Overall, by 5 dpi and afterward, the mean rectal temperatures in Group 3 were significantly higher than those in Group 2. No significant difference arose in the mean rectal temperatures between Groups 1, 2, and 4.
Antibody to PRRSV
Antibodies against PRRSV were observed in Groups 1 and 2 beginning at 7 days after EDRD1 inoculation in some pigs and were over the cutoff line (sample-to-positive ratio, S/P, 0.4) in all animals at 11 days after EDRD1 inoculation (−15 dpi of the challenge) (Fig. 3). Although the S/P ratio of four of five pigs in Group 2 was 1.28, 1.24, 1.37, and 1.32, that of pig G12 was 0.63 at 11 days after EDRD1 inoculation. The S/P ratio of G12 then gradually rose very slowly compared with those of the other four pigs, and that of pig G12 was 0.92 at 0 dpi. In Group 3, the positive conversion of antibody to PRRSV was observed at 7 or 10 dpi, and the S/P ratio of viral antibody gradually rose. By contrast, no antibodies against PRRSV were observed in uninoculated Group 4.
Serum viral load (viral RNA copies/mL) in Group 3 was significantly higher than that of Group 2 at 3 and 13 dpi (Fig. 4A). The mean 10186-614 viral RNA in Group 3 pigs sharply rose at 3 and 7 dpi and plateaued afterward between 2.8 × 106 and 4.9 × 106 copies/mL. By contrast, the viral load in two of five pigs in Group 2 transiently rose at 7 dpi (pig G9) and 7 and 10 dpi (pig G7) and in a third pig reached 5.3 × 103 to 2.9 × 104 copies/mL from 7 to 13 dpi (pig G12). For the remaining two pigs in Group 2, viral RNA in both sera and oral fluid remained under the detection limit (pigs G8 and G10) after 10186-614 challenge. Viral RNA was detected in oral fluid from all animals in Group 3 at 7, 10, and 13 dpi (Fig. 4B). The viral load in the oral fluid in Group 2 was transiently detected in pigs G7 and G9 at 7 dpi and was intermittently detected in pig G12 at 7, 10, and 13 dpi. At necropsy on 16 dpi, viral RNA was detected in all tested organs, namely, the lung, liver, tonsil, kidney, spleen, and brain of all pigs inoculated with 10186-614 in Group 3 (Fig. 5A). Mean viral RNA copies/g tissue were approximately 5.2 × 106 in the lung, 2.2 × 105 in the liver, 1.3 × 107 in the tonsil, 2.6 × 105 in the kidney, 7.1 × 105 in the spleen, and 1.3 × 104 in the brain. By contrast, viral RNA was detected from only the tonsils (2.3 × 107 copies/g) and spleen (1.0 × 106 copies/g) of pig G12 in Group 2 (Fig. 5B). Expectedly, no 10186-614 viral RNA was found in the sera, oral fluid, or any tissues of pigs in Groups 1 and 4.
Gross and histological lesions
Gross anatomic observation after euthanization on 16 dpi revealed prominent lesions of pneumonia and thymic atrophy in the organs of Group 3 pigs. The lung lesions in these pigs were characterized by dark-reddish pneumonia with congested edematous consolidation in the whole lobes. (Fig. 6C, upper panel). By contrast, the lungs in most pigs of Group 2 were only slightly red compared with those in Group 1. Mean (standard deviation, SD) gross lung lesion scores were 0.8% ± 1.8% in Group 1, 12.0% ± 8.9% in Group 2, 77.2% ± 14.8% in Group 3, and 0% ± 0% in Group 4, with a significant difference between Groups 2 and 3 (Fig. 6A).
Histopathological pneumonic lesions in Group 3 were characterized by severe interstitial pneumonia with marked accumulation of proteinaceous and cell debris in alveolar spaces, marked septal infiltration with macrophages and capillary congestion in the alveolar wall (Fig. 6C, lower panel). By contrast, histopathological pneumonic lesions in Group 2 were characterized by slight to moderate histiolymphocytic infiltration in the alveolar wall. The microscopic pneumonic score (mean ± SD) in Group 2 (1.1 ± 0.3) was significantly lower than that in Group 3 (3.3 ± 0.4) (Fig. 6B).