Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 in vivo. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2-MP120 had decreased virulence, eliciting weight gains, body temperatures, and histopathologic lesions similar to those in the negative control group. However, compared to CA-2-P100 infection, CA-2-MP120 yielded consistently higher viremia kinetics and enhanced antibody responses in pigs. All pigs inoculated with CA-2-MP120 developed viremia and seroconverted to PRRSV. During 20 passages in PAM cells, CA-2-MP120 acquired 15 amino acid changes that were mostly distributed in nsp2 and minor structural protein-coding regions. Among these changes, 6 mutations represented reversions to the sequences of the reference CA-2 and parental CA-2-P20 strains. These genetic drifts may be hypothetical molecular markers associated with PRRSV macrophage tropism and virulence. Our results indicate that the PAM-passaged CA-2-MP120 strain is a potential candidate for developing a live, attenuated PRRSV vaccine.

Keywords: attenuated vaccines; macrophage tropism; porcine reproductive and respiratory syndrome virus; virulence; whole genome sequencing.

Fig. 1. Average body temperature (A) and average daily weight gain (B) of pigs after challenge with the CA-2-P20, CA-2-P100, or CA-2-MP120 strains of porcine reproductive and respiratory syndrome virus or mock challenge with culture medium. Rectal temperatures equal to or greater than 40℃ were defined as fever. Error bars represent SD. “a” denotes a significant difference (p < 0.05) between the CA-2-P20 and CA-2-P100 groups; “b” denotes a significant difference (p < 0.05) between the CA-2-P20 and CA-2-MP120 groups.

Fig. 2. Virus replication in vivo and seroconversion. (A) Mean levels of viremia of pigs infected with CA-2-P20, CA-2-P100, or CA-2-MP120. TCID₅₀, 50% tissue culture infectious dose. (B) Virus-specific antibody response of pigs measured by using a commercial enzyme-linked immunosorbent assay kit. Serum samples were considered positive for antibodies to porcine reproductive and respiratory syndrome virus if the sample:positive (S/P) ratio was equal to or greater than 0.4. Error bars represent SD. “a” denotes a significant difference (p < 0.05) between the CA-2-P20 and CA-2-P100 groups; “b” denotes a significant difference (p < 0.05) between the CA-2-P20 and CA-2-MP120 groups; “c” denotes a significant difference (p < 0.05) between the CA-2-P100 and CA-2-MP120 groups.

Fig. 3. Photomicrographs of the lungs and lymph nodes (LN) from pigs in the CA-2-P20, CA-2-P100, CA-2-MP120, and control groups. H&E-stained tissues from the pigs in each group were histopathologically examined. 100×. The upper panels represent lung sections from pigs of each group. A CA-2-P20-infected pig (Group 1) shows a marked interstitial pneumonia with alveolar walls thickened by massive lymphocyte and macrophage infiltration surrounding perivascular cuffing (arrows), whereas pigs inoculated with CA-2-P100 (Group 2) or CA-2-MP120 (Group 3) exhibit normal lung lesions comparable to control pig lung. The lower panels represent LN sections from pigs of each group. A pig in Group 1 displayed lymphoid follicular hyperplasia (arrow), whereas pigs in Groups 2 and 3 exhibited no remarkable LN lesions, as was observed in control pig LN samples.

Fig. 4. Average histopathologic scores of lung (A) and lymph node (B) tissues from pigs in the CA-2-P20, CA-2-P100, CA-2-MP120, and control groups. The results are expressed as mean values from 6 pigs in each group, and error bars represent SD. ^*p = 0.001–0.05, ^**p < 0.001.

Fig. 5. Schematic representation of amino acid (aa) differences between the parental CA-2-P20 virus and its cell-adapted derivatives. The genomic sequences of the virus strains were determined as described in Table 1. The organization of the CA-2 genome (approximately 15 kb) is shown at the top. The coding regions for each nonstructural protein (nsp) in open reading frames (ORFs) 1a and 1b and for each structural protein are indicated. The lower panels show the genomes of the parental CA-2-P20 strain and its cell-adapted derivatives, where the vertical lines (black) represent a 1 aa substitution relative to the predicted aa sequence of CA-2-P20. The vertical dotted lines denote a 1 aa substitution in the overlapping coding regions for nsp2/nsp2TF, GP2/E, and GP5/ORF5a (silent mutation in nsp2, whereas a non-silent mutation in nsp2TF; silent mutation in GP2, whereas a non-silent mutation in E; non-silent mutation in GP5, whereas a silent mutation in ORF5a). The vertical lines (red) in the PAM-passaged CA-2-MP-120 strain indicate 15 aa changes relative to the predicted aa sequence of CA-2-P100 and the vertical arrows (red) indicate reverting mutations that restore the sequence of CA-2-P20.