The Junction Between nsp1β and nsp2 in the Porcine Reproductive and Respiratory Syndrome Virus Genome Is a New Site for the Insertion and Expression of Foreign Genes

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered a promising viral vector for the expression and delivery of foreign genes for the development of a new generation of multi-valent vaccines against PRRSV and other porcine viruses, as well as for analyses of the immune response against PRRSV and anti-PRRSV component screening. In the present study, the junction site between nsp1β and nsp2 in the PRRSV genome was tested for the insertion and expression of foreign genes. Three foreign genes, including eGFP, iLOV3, and TEVp, were inserted into the intergenic junction between nsp1β and nsp2 and expressed by the respective recombinant PRRSVs (rPRRSV-SH01-eGFP, rPRRSV-SH01-iLOV3, and rPRRSV-SH01-TEVp) in vitro in mammalian cells. Analysis of the growth kinetics of the rescued recombinant PRRSVs showed no significant differences between the recombinant PRRSVs and their parental viruses. The inserted genes were consistently present in the viral genome during serial passage in vitro (for at least 20 passages). In addition, rPRRSV-SH01-eGFP can be used as a reporter virus for rapid detection of neutralizing antibodies against PRRSV through a fluorescent focus unit reduction-based assay. These data demonstrate that the junction between nsp1β and nsp2 is a new site that is suitable for the insertion and expression of foreign genes, providing a new option to express and deliver foreign genes using PRRSV-based vectors for different purposes, such as the development of multi-valent vaccines against PRRSV and other porcine viruses.

Keywords

• porcine reproductive and respiratory syndrome virus
• viral vector
• recombinant virus
• nsp1β
• nsp2
• foreign gene expression