Moe Key Laboratory of Aquatic Food Safety/State Key Laboratory for Bio-control, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.
Virus Res. 2011 Nov;161(2):140-9. doi: 10.1016/j.virusres.2011.07.016. Epub 2011 Aug 4.
A new continuous cell line (KCF-1) from caudal fin of koi, Cyprinus carpio koi, was developed and sub-cultured more than 100 passages since the present study was initiated in March 2006. KCF-1 predominantly consisted of short fibroblast-like cells and grew well in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Chromosome analysis revealed that 56% of the KCF-1 cells maintained normal diploid chromosome number (2n=100) at Passage 82. Using the KCF-1 cell line, a strain of cyprinid herpesvirus 3 (designated as CyHV-3-QY08) was isolated from the diseased koi. CyHV-3-QY08 continuously propagated in the KCF-1 cells, as confirmed by immunofluorescence assay (IFA) and transmission electron microscopy (TEM). KCF-1 cells infected with CyHV-3-QY08 produced typical cytopathic effects characterized by severe vacuolation, deformation of nuclei, and marginalization of the nuclear chromatin, which are consistent with those of previous reports. CyHV-3-QY08 was purified and subsequently analyzed by SDS-PAGE and TEM. The results showed that the purified virions contained two types of morphologies and were composed of more than 30 obvious viral polypeptides. An infectivity experiment revealed that CyHV-3-QY08 could cause 100% mortality in the infected koi. Based on the genome sequence of CyHV-3-I/U, the CyHV-3(I/U)-ORF136 homologue in CyHV-3-QY08 was cloned and sequenced. Multiple sequence alignments of CyHV-3-I/U-ORF136 homologues showed that CyHV-3-QY08 belonged to the typical Asian genotype. The CyHV-3(I/U)-ORF136 homologue seems to be a novel molecule marker, which can be used to distinguish Asia isolates from Europe-America strains.
自 2006 年 3 月本研究开始以来，已从锦鲤（Cyprinus carpio koi）尾鳍中开发并传代培养了超过 100 代的新连续细胞系（KCF-1）。KCF-1 主要由短成纤维细胞样细胞组成，在补充有 10％胎牛血清（FBS）的 Dulbecco 改良 Eagle 培养基（DMEM）中生长良好。染色体分析显示，KCF-1 细胞的 56％在第 82 代时保持正常的二倍体染色体数（2n=100）。使用 KCF-1 细胞系，从患病锦鲤中分离出了一种鲤鱼疱疹病毒 3 株（命名为 CyHV-3-QY08）。CyHV-3-QY08 在 KCF-1 细胞中连续繁殖，通过免疫荧光法（IFA）和透射电子显微镜（TEM）确认。感染 CyHV-3-QY08 的 KCF-1 细胞产生了典型的致病变效应，其特征是严重的空泡化，细胞核变形和核染色质边缘化，与先前的报道一致。纯化后的 CyHV-3-QY08 经 SDS-PAGE 和 TEM 分析。结果表明，纯化的病毒粒子包含两种形态，由 30 多种明显的病毒多肽组成。感染实验表明，CyHV-3-QY08 可导致感染锦鲤的 100％死亡率。根据 CyHV-3-I/U 的基因组序列，克隆并测序了 CyHV-3-QY08 中的 CyHV-3（I/U）-ORF136 同源物。CyHV-3-I/U-ORF136 同源物的多重序列比对表明，CyHV-3-QY08 属于典型的亚洲基因型。CyHV-3（I/U）-ORF136 同源物似乎是一种新型分子标记，可用于区分亚洲分离株和欧洲-美洲株。
