Division of Zoonoses, State Key Laboratory of Special Economic Animal Molecular Biology, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, Jilin 130122, PR China.
Vet Microbiol. 2012 Dec 28;161(1-2):58-65. doi: 10.1016/j.vetmic.2012.07.011. Epub 2012 Jul 17.
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 β-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 TCID(50) 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.
猪繁殖与呼吸综合征（PRRS）是猪生产地区一种具有重要经济意义的疾病。已经开发了许多疫苗策略来控制该疾病，但没有一种策略完全成功。因此，开发能够大量生产 PRRSV 疫苗的细胞系是非常必要的。为了确定 Nsp2 在 MARC-145 细胞中高致病性猪繁殖与呼吸综合征病毒（HP-PRRSV）复制中的作用，我们使用基于 RNA 干扰的 Nsp2 短发夹 RNA 构建了表达 HP-PRRSV Nsp2 基因的细胞系。我们使用保守的 HP-PRRSV Nsp2 序列设计了短干扰 RNA，并测试了它们在体外细胞转染中沉默 PRRSV 转录表达和复制的能力。使用半定量实时 PCR 确定 Nsp2、ORF7 和 β-肌动蛋白 mRNA 的表达。发现针对 Nsp2 的 siRNA 感染可降低 PRRSV 感染的 MARC-145 细胞中的 Nsp2 表达。通过 G418 筛选 MARC-145-TJ Nsp2 和 MARC-145-TJM Nsp2 细胞系，用 HP-PRRSV 感染，用正常的 MARC-145 细胞mock 感染，然后在 CPE 出现后用 TCID(50)计算病毒滴度。Nsp2 的下调诱导 PRRSV 复制的显著减少，导致结构蛋白减少。靶向 Nsp2 的 siRNA 下调了 MARC-145 细胞中 Nsp2 的表达，并诱导 PRRSV 在 MARC-145 细胞中的复制减少。shRNA 载体 S-1 和 S-2 可有效诱导 MARC-145 中的病毒复制抑制。结果表明，表达高致病性 PRRSV TJ 和减毒 TJM Nsp2 基因的细胞仍然稳定。这些细胞中的 PRRSV 复制速度比 MARC-145 细胞更快，尤其是在早期阶段。这表明 Nsp2 在 PRRSV 增殖中起积极作用。
