A transgenic Marc-145 cell line of piggyBac transposon-derived targeting shRNA interference against porcine reproductive and respiratory syndrome virus

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is now considered to be one of the most important diseases in countries with intensive swine industries. The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In this study, we designed 5 of the small hairpin RNAs (shRNAs) targeting the GP5 and M gene of PRRSV respectively, and investigated their inhibition to the production of PRRSV. The highest activity displayed in shRNAs of the ORF6e sequence (nts 261-279), which the inhibition rate reached was 99.09%. The result suggests that RNAi technology might serve as a potential molecular strategy for PRRSV therapy. Furthermore, the transgenic Marc-145 cell line of piggyBac transposon-derived targeting shRNA interference against PRRS virus was established. It presented stable inhibition to the replication and amplification of PRRS. The work implied that shRNAs targeting the GP5 and M gene of PRRSV may be used as potential RNA vaccines in vivo, and supplied the screening methods of transformed pig embryonic fibroblast which are prerequisite for the disease-resistant transgenic pigs to PRRS.

RNAi-based inhibition of porcine reproductive and respiratory syndrome virus replication in transgenic pigs

“…A recent report has declared that siRNA transgenesis could confer viral infection resistance to pseudorabies virus (PRV) on mice (Daniel-Carlier et al, 2013). Although it has been shown that transient delivery of siRNA by targeting viral genes protects susceptible cell lines (He et al, 2007), stable transgenic cell line presented stable inhibition to virus (Zhou et al, 2012), and recombinant adenovirusmediated RNAi guards animals against viral challenges , it remains unclear whether transgenesis of siRNA in pigs can confer permanent resistance against PRRSV infection. Previously, we designed siRNAs to target the open reading frame (ORF) 1b and 6 regions of PRRSV and showed that siRNA-1B372 targeting ORF1b had the strongest virus-inhibiting effect in MARC-145 cells.…”

Short hairpin RNAs targeting M and N genes reduce replication of porcine deltacoronavirus in ST cells

“…selectively inactivates the mRNA of the target gene. RNAi has been successfully used in infection inhibition studies of animal viruses, such as porcine epidemic diarrhea virus (PEDV) [15], influenza virus A [16], porcine transmissible gastroenteritis virus (TGEV) [17], and porcine reproductive and respiratory syndrome virus [18]. However, whether RNAi inhibits the replication of PDCoV has not been reported.…”