Role of non-structural protein 2 in the regulation of the replication of the porcine reproductive and respiratory syndrome virus in MARC-145 cells: Effect of gene silencing and over expression

摘要

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas. Many vaccine strategies have been developed to control the disease, but none have yet been completely successful. The development of a cell line that can produce large yields of PRRSV vaccine is very necessary. In order to determine the role of Nsp2 in the replication of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in MARC-145 cells, we used an RNA interference-based short hairpin RNA of Nsp2 and constructed cell lines expressing the HP-PRRSV Nsp2 gene. Conserved HP-PRRSV Nsp2 sequences were used to design short interfering RNAs and test their ability to silence PRRSV transcript expression and replication in cells in vitro transfection. Nsp2, ORF7, and beta-actin mRNA expression were determined using semi-quantitative real-time PCR. Infection with siRNA targeting Nsp2 was found to reduce the Nsp2 expression in MARC-145 cells infected with PRRSV. Both MARC-145-TJ Nsp2 and MARC-145-TJM Nsp2 cell lines were screened by G418, which were infected with HP-PRRSV, normal MARC-145 cells for mock, and then virus titers were calculated by TCID50 after the CPE showing up. The downregulation of Nsp2 induced a remarkable decrease in PRRSV replication, causing the reduction of structural protein. The Nsp2-targeted siRNA was found to downregulate the expression of Nsp2 in MARC-145 cells and inducing replication reduce of PRRSV in MARC-145 cells. The shRNA vectors S-1 and S-2 could effectively induce the inhibition of viral replication in MARC-145. Results showed that cells expressing the Nsp2 gene of the highly pathogenic PRRSV TJ and attenuated TJM remained stable. PRRSV replication was faster in these cells than in MARC-145 cells, especially during the early stage. This shows that Nsp2 plays a positive role in PRRSV proliferation. (C) 2012 Elsevier B.V. All rights reserved.