Choongang Vaccine Laboratory, Daejeon 304-348, Republic of Korea.
School of Life Sciences, KNU Creative BioResearch Group (BK21 plus program), Kyungpook National University, Daegu 702-701, Republic of Korea.
Vet Microbiol. 2014 Jun 4;170(3-4):232-45. doi: 10.1016/j.vetmic.2014.02.027. Epub 2014 Mar 4.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine virus that exhibits genetic and pathogenic heterogeneity among isolates. The present study was conducted to determine the complete genome sequence and pathogenicity of two Korean type 2 PRRSV nonstructural protein 2 (nsp2) deletion mutants, CA-2 and KNU-12-KJ4. The full-length genomes of CA-2 and KNU-12-KJ4 were determined to be 15,018 and 15,019 nucleotides in length, excluding the poly(A) tail, respectively, which were 393- or 392-nucleotide shorter than that of the type 2 NA prototype strain VR-2332 due to the presence of notable large deletions within the nsp2 gene. The genomes of CA-2 and KNU-12-KJ4 consisted of a 189- or 190-nucleotide 5' untranslated region (UTR), a 14,677-nucleotide protein-coding region, and a 151-nucleotide 3' UTR. Whole genome evaluation revealed that the nucleotide sequences of CA-2 and KNU-12-KJ4 are most similar to each other (10.7% sequence divergence), and then to the Korean strain CA-1 (11.3% sequence divergence) and the US strain MN184C (13.1% sequence divergence), respectively. To evaluate the in vitro immunity of nsp2 deletion variants, we sought to explore alteration of inflammatory cytokine and chemokine expression in PAM-pCD163 cells infected with each virus strain using quantitative real-time RT-PCR. Cytokine genes including IL-8, IL-10, and TNF-α, and chemokines such as MCP-1 and RANTES were found to be significantly elevated in nsp2 deletion virus-infected PAM cells. In contrast, expression of interferons (IFN-β, γ, and λ) and antiviral genes including ISG-15, -54, and -56 were unchanged or down-regulated in PAM cells infected with the nsp2 deletion mutants. Animal studies to assess the pathogenicity of nsp2 deletion PRRSVs demonstrated that both CA-2 and KNU-12-KJ4 strains notably produce weight loss in infected pigs. Furthermore, the nsp2 deletion mutants replicated well in pigs with significantly increased and prolonged viremia kinetics. Taken together, our results indicate that, among the three isolates, the outcome of in vitro and in vivo infection by CA-2 and KNU-12-KJ4 is comparable, suggesting that the large nsp2 deletion may be one of the viral genetic determinants contributing to PRRSV pathogenicity.
猪繁殖与呼吸综合征病毒（PRRSV）是一种在全球范围内普遍存在的猪病毒，其分离株存在遗传和致病性的异质性。本研究旨在确定两种韩国 2 型 PRRSV 非结构蛋白 2（nsp2）缺失突变体 CA-2 和 KNU-12-KJ4 的完整基因组序列和致病性。CA-2 和 KNU-12-KJ4 的全长基因组分别为 15018 和 15019 个核苷酸，不包括 poly(A)尾巴，由于 nsp2 基因内存在显著的大缺失，它们比 2 型 NA 原型株 VR-2332 短 393-或 392 个核苷酸。CA-2 和 KNU-12-KJ4 的基因组由一个 189-或 190 个核苷酸的 5'非翻译区（UTR）、一个 14677 个核苷酸的蛋白编码区和一个 151 个核苷酸的 3'UTR 组成。全基因组评估显示，CA-2 和 KNU-12-KJ4 的核苷酸序列彼此最为相似（10.7%序列差异），然后与韩国株 CA-1（11.3%序列差异）和美国株 MN184C（13.1%序列差异）最为相似。为了评估 nsp2 缺失变体的体外免疫，我们试图通过定量实时 RT-PCR 研究感染每种病毒株的 PAM-pCD163 细胞中炎性细胞因子和趋化因子表达的变化。结果发现，IL-8、IL-10 和 TNF-α 等细胞因子基因和 MCP-1 和 RANTES 等趋化因子在 nsp2 缺失病毒感染的 PAM 细胞中显著升高。相比之下，感染 nsp2 缺失突变体的 PAM 细胞中干扰素（IFN-β、γ 和 λ）和抗病毒基因（ISG-15、-54 和 -56）的表达不变或下调。评估 nsp2 缺失 PRRSV 致病性的动物研究表明，CA-2 和 KNU-12-KJ4 株均显著导致感染猪体重减轻。此外，nsp2 缺失突变体在猪体内复制良好，病毒血症动力学显著增加且持续时间延长。综上所述，我们的结果表明，在这三种分离株中，CA-2 和 KNU-12-KJ4 的体外和体内感染结果相当，这表明大的 nsp2 缺失可能是 PRRSV 致病性的病毒遗传决定因素之一。
