Insertion and deletion in a non-essential region of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome (PRRS) virus: effects on virulence and immunogenicity

Abstract

Developing a vaccine that can differentiate infected and vaccinated animals (DIVA) is a new challenge in the design of a vaccine for porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein 2 (nsp2) is the single largest viral product, and it has multiple roles in polypeptide processing and replication complex formation. Using reverse genetics and an infectious PRRSV cDNA clone, we constructed several deletion mutants in the non-essential region of nsp2. One mutant, which has a 131 amino acid deletion within a relatively conserved region of nsp2, was recovered and found to produce a viable virus. The deleted region was replaced with a peptide tag encoding eight amino acids. A recombinant virus containing the 131 amino acid deletion was found to produce normal virus yields in MARC-145 cells and porcine alveolar macrophages (PAM); however, gross and micro-histopathology showed that the virus was less virulent in pigs. The 131 amino acid peptide was expressed as a recombinant protein and used to coat enzyme-linked immunosorbent assay (ELISA) plates. This peptide was recognized by sera from pigs infected with wild-type virus, but not by sera from pigs infected with the deletion mutant. The results from this study show that nsp2 is an important target for the development of marker vaccines and for virus attenuation.

Cytokine profiles and phenotype regulation of antigen presenting cells by genotype-I porcine reproductive and respiratory syndrome virus isolates

“…Also, it shows that IL-10 seems to play an important role in SLA-I, CD14 and probably CD80/86 but it is evident that this cytokine is not the only element in that regulation and, as some reports indicate [31,32] non-structural proteins seem to play an important role in the regulation of the innate responses against PRRSV. From a practical point of view, the present results also suggest that immunological studies of PRRSV cannot be performed with a single PRRSV strain if a global vision is desired or that genetic variability of PRRSV has to be taken into account when using clones or in vitro models.…”

Antiviral Regulation in Porcine Monocytic Cells at Different Activation States

“…Animal procedures and the isolation of alveolar macrophages and peripheral blood mononuclear cells (PBMCs) were conducted as described previously (29,(31)(32)(33)(34)(35)(36)(37). PBMCs were isolated using a 60% Ficoll-Paque Plus gradient (GE Healthcare, Piscataway, NJ).…”

Characterization of a serologic marker candidate for development of a live-attenuated DIVA vaccine against porcine reproductive and respiratory syndrome virus

“…Therefore, the most feasible approach to develop a live-attenuated DIVA vaccine against PRRSV would be selectively eliminating a small protein fragment or an epitope, instead of deleting an entire protein. Thus far, efforts to develop a DIVA vaccine for PRRSV have been focused on deleting different immunogenic fragments of the viral nsp2 because this protein can tolerate large deletions [41–43]. However, the biggest shortcoming of the nsp2-mutant viruses, in the context of a DIVA vaccine, is that the differential peptide-based ELISA used in conjunction with the DIVA vaccine has very limited diagnostic sensitivity [42], mainly due to the substantial genetic variation of the nsp2 [44,45].…”