Stable expression of foreign gene in nonessential region of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus: applications for marker vaccine design.

The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to be highly heterogeneous and variable among PRRSV strains and some sequences in the middle region of the nsp2 are not essential to viral replication. Recent studies have attempted to insert foreign genes in the nsp2 nonessential regions but the foreign genes were not stably expressed by recombinant viruses in vitro. In the present study, we first constructed an infectious cDNA clone with deletion of 75 nucleotides (25 amino acids) in the nsp2 region (rHuN4-F112-Δ508-532) of the attenuated vaccine virus HuN4-F112 derived from a highly pathogenic PRRSV HuN4 and then inserted a gene fragment encoding a immunodominant B-cell epitope (49 amino acids) of Newcastle disease virus (NDV) nucleoprotein (NP) in-frame into the deletion site. The viable recombinant virus was rescued from the full-length cDNA infectious clone in vitro. The engineered viruses rescued from the cDNA clone indicated that the deletions of 75 nucleotides and insertion of NDV NP gene in the nsp2 region did not affect viral replication; they had similar growth kinetics to its parental virus. The inserting gene could be expressed consistently when the recombinant virus was passaged up to twenty times in cell cultures as determined by immunofluorescence assay (IFA) and genomic sequencing. To investigate the potential application of the NDV NP gene-inserted PRRSV as a marker vaccine, piglets were immunized with the recombinant virus and then challenged with lethal dose of highly pathogenic PRRSV. The immunized piglets produced specific antibodies against both the NDV NP and PRRSV, and lacked antibodies against the deleted 25aa nsp2 epitope. After challenge, all immunized piglets were protected from clinical disease or death, while all piglets in control group died (5/5) by ten days post challenge. The results of the present study indicated that the recombinant PRRSV (rHuN4-F112-Δ508-532) could be used as a potential marker vaccine against PRRS.