Effect of an 88-amino-acid deletion in nsp2 of porcine reproductive and respiratory syndrome virus on virus replication and cytokine responses in vitro

Abstract

Previously, a spontaneous 88-amino-acid (aa) deletion in nsp2 was associated with cell-adaptation of porcine reproductive and respiratory syndrome virus (PRRSV) strain JXM100, which arose during passaging of the highly pathogenic PRRSV (HP-PRRSV) strain JX143 in MARC-145 cells. Here, to elucidate the biological role of this deletion, we specifically deleted the region of a cDNA clone of HP-PRRSV strain JX143 (pJX143) corresponding to these 88 amino acids. The effect of the deletion on virus replication in cultured cells and transcriptional activation of inflammatory cytokines and chemokines in pulmonary alveolar macrophages (PAMs) was examined. Mutant virus with the 88-aa deletion in nsp2 (rJX143-D88) had faster growth kinetics and produced larger plaques in MARC-145 cells than the parental virus (rJX143), suggesting that the deletion enhanced virus replication in MARC-145 cells. In contrast, the overall yield of rJX143 was almost 1 log higher than that of rJX143-D88, suggesting that the 88-aa deletion in nsp2 decreased the production of infectious viruses in PAMs. Infection with the mutant virus with the 88-aa deletion resulted in increased mRNA expression of type I interferon (IFN-α and IFN-β) and chemokines genes. In addition, the mRNA expression of antiviral genes (ISG15, ISG54 and PKR) regulated by the IFN response was upregulated in PAMs infected with the mutant virus rJX143-D88. Our results demonstrate that virus-specific host immunity can be enhanced by modifying certain nsp2 epitope regions. These findings provide important insights for understanding virus pathogenesis and development of future vaccines.

Generation of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing Two Marker Genes

“…Interestingly, spontaneous amino acid deletions in nsp2 have also occurred during serial passages of PRRSV in MARC-145 cells (20,23,35). Some studies also showed that engineered deletion of a long coding region in the HVR of nsp2 is dispensable for virus viability (21)(22)(23). In the present study, we constructed a recombinant PRRSV clone harboring a 465-nucleotide deletion in the HVR of nsp2 that had previously occurred naturally in a PRRSV strain isolated in our laboratory (Genbank No.…”

“…Natural spontaneous amino acids deletions and insertions were found in the hypervariable region (HVR) of nsp2 (15)(16)(17)(18)(19). The HVR of nsp2 can also be engineered for the insertion of foreign genes or deletion of specific individual immune dominant epitopes in order to develop recombinant marker PRRSV vaccines (8,17,(20)(21)(22)(23)(24). However, fusion of the foreign genes within the HVR of nsp2 in most recombinant viruses does not always maintain stability of the resultant recombinant viruses or sometimes there is a loss of biological activity of the target genes after serial passages in MARC-145 cells (8,22,23).…”