Gong Wenjie, Lu Zongji, Zhang Li, Xie Xiaoming, Jiang Daliang, Jia Junjie, Guo Huancheng, Shi Jishu, Tu Changchun
Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, 130122, China.
Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, 66506, USA.
Virus Genes. 2016 Oct;52(5):651-9. doi: 10.1007/s11262-016-1350-x. Epub 2016 May 7.
Classical swine fever (CSF) still causes substantial economic losses in the pig industry in China. This study reports the isolation and characterization of a field CSF virus named GD53/2011 from pig kidney tissue collected during a CSF outbreak in Guangdong province, China. Phylogenetic analysis based on the full-length E2 gene sequence revealed that this isolate belongs to CSFV sub-subgenotype 2.1c. To further understand the replication characteristics, GD53/2011 was subsequently adapted in PK-15 cells, and its full-length genome was sequenced. After adaptation in PK-15 cells, the titer of GD53/2011 was significantly increased from 10(3.39) TCID50/ml at passage 6 (F6) to 10(8.50) TCID50/ml at passage 46 (F46) with the peak titer obtained at 48 h post-inoculation. Sequence comparison revealed that the E(rns) gene at passages 6, 15, and 25 of GD53/2011 was identical to that in the original tissue, but one amino acid substitution (S476R) was detected at passages 35 and 46. Furthermore, E2 gene sequences at passages 6, 15, 25, 35, and 46 was found identical to that in the original tissue, indicating that the E2 gene was stable during CSF virus adaptation in PK-15 cells. Full-length protein sequence comparison of GD53/2011 with other 2.1 sub-subgenotype isolates showed that Core and NS5A, rather than E2, are more genetically variable. Taken together, a field CSFV strain GD53/2011 was isolated, fully sequenced, and adapted to high growth titer in PK-15 cells, which might be suitable for future studies on CSFV infection, replication, and vaccine development.
经典猪瘟（CSF）在中国养猪业中仍造成巨大经济损失。本研究报告了从中国广东省一次CSF疫情期间采集的猪肾组织中分离并鉴定出一株名为GD53/2011的CSF野毒株。基于全长E2基因序列的系统发育分析表明，该分离株属于CSFV亚亚基因型2.1c。为进一步了解其复制特性，随后将GD53/2011在PK - 15细胞中传代适应，并对其全长基因组进行测序。在PK - 15细胞中传代适应后，GD53/2011的滴度从第6代（F6）时的10(3.39) TCID50/ml显著提高到第46代（F46）时的10(8.50) TCID50/ml，接种后48小时达到滴度峰值。序列比较显示，GD53/2011在第6、15和25代时的E(rns)基因与原始组织中的相同，但在第35和46代时检测到一个氨基酸替换（S476R）。此外，发现第6、15、25、35和46代时的E2基因序列与原始组织中的相同，表明E2基因在CSF病毒在PK - 15细胞中适应过程中是稳定的。GD53/2011与其他2.1亚亚基因型分离株的全长蛋白序列比较表明，Core和NS5A而非E2在遗传上更具变异性。综上所述，分离出了一株CSFV野毒株GD53/2011，对其进行了全序列测定，并使其在PK - 15细胞中适应至高生长滴度，这可能适用于未来关于CSFV感染、复制和疫苗开发的研究。
