Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061-0913 , USA.
Virus Res. 2011 Sep;160(1-2):264-73. doi: 10.1016/j.virusres.2011.06.027. Epub 2011 Jul 7.
A highly pneumovirulent strain of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was isolated from a pig exhibiting typical PRRS in the early 90s. While passaging the virus in monkey kidney cells, we identified a large spontaneous deletion of a 435-bp in the nsp2 gene. To assess the biological significance of this spontaneous deletion, we first determined the full-length genomic sequence of this virus and established a DNA-launched infectious clone of the passage 14 virus containing the 435-bp nsp2 deletion (designated as pIR-VR2385-CA). The full-length viral genome engineered with two ribozyme elements at both ends was placed under the control of the eukaryotic CMV promoter. The infectious virus was successfully rescued from pIR-VR2385-CA DNA-transfected BHK-21 cells. To characterize the biological and pathological significance of this large nsp2 deletion, we subsequently constructed another DNA-launched infectious clone, pIR-VR2385-R, in which we restored the deleted 435-bp nsp2 sequence back to the pIR-VR2385-CA backbone. The growth characteristics of the two rescued viruses (VR2385-CA and VR2385-R) were compared, and the results showed that the VR2385-CA virus with the nsp2 deletion replicated more efficiently in vitro (1.0-1.5 log titer higher) than the VR2385-R virus with the restored nsp2 sequence but the VR2385-CA virus exhibited a significantly reduced serum viral RNA load in vivo. A comparative pathogenicity study in pigs (n=10) revealed that the nsp2 deletion had no effect on virus virulence, and the restored nsp2 sequence in the VR2385-R virus remains stable during virus replication in pigs. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no effect on the pathogenicity of the virus.
从 90 年代初期表现出典型 PRRS 症状的猪体中分离到一株高致病性猪繁殖与呼吸综合征病毒（PRRSV），其毒株为 ATCC VR2385。在对该病毒进行猴肾细胞传代的过程中，我们发现 nsp2 基因发生了一个 435bp 的自发缺失。为了评估该自发缺失的生物学意义，我们首先确定了该病毒的全长基因组序列，并构建了一个包含 nsp2 缺失（命名为 pIR-VR2385-CA）的第 14 代病毒的 DNA 启动感染性克隆。该全长病毒基因组在两端各带有两个核酶元件，受真核 CMV 启动子调控。该感染性病毒成功地从 pIR-VR2385-CA DNA 转染的 BHK-21 细胞中拯救出来。为了阐明该 nsp2 缺失的生物学和病理学意义，我们随后构建了另一个 DNA 启动的感染性克隆 pIR-VR2385-R，其中我们将缺失的 435bp nsp2 序列恢复到 pIR-VR2385-CA 骨架上。比较了两个拯救病毒（VR2385-CA 和 VR2385-R）的生长特征，结果表明，缺失 nsp2 的 VR2385-CA 病毒在体外的复制效率更高（滴度高出 1.0-1.5 个对数），而恢复 nsp2 序列的 VR2385-R 病毒在体内的血清病毒 RNA 载量则显著降低。在猪（n=10）中的比较致病性研究表明，nsp2 缺失对病毒毒力没有影响，并且在猪体内病毒复制过程中，VR2385-R 病毒中的恢复 nsp2 序列保持稳定。该研究结果表明，nsp2 缺失在体外增强了 PRRSV 的复制，但对病毒的致病性没有影响。
