Characterization of a new cell line from caudal fin of koi, Cyprinus carpio koi, and first isolation of cyprinid herpesvirus 3 in China
Characterization of a new cell line from caudal fin of koi, Cyprinus carpio koi, and first isolation of cyprinid herpesvirus 3 in China
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DOI：
10.1016/j.virusres.2011.07.016
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发表时间：
2011-11-01
期刊：
影响因子：
5
通讯作者：
He, Jianguo
中科院分区：
文献类型：
--
作者：
Dong, Chuanfu;Weng, Shaoping;He, Jianguo
本研究自2006年3月启动以来，从锦鲤（Cyprinus carpio koi）尾鳍中培育出一株新的连续细胞系KCF-1，并进行了100多次传代培养。KCF-1主要由短成纤维细胞样细胞组成，在添加10%胎牛血清的Dulbecco's modified Eagle培养基（DMEM）中生长良好。染色体分析显示，在82代时，56%的KCF-1细胞维持正常的二倍体染色体数（2n = 100）。利用KCF-1细胞系，从病锦鲤中分离出一株CyHV-3-QY08。免疫荧光法（IFA）和透射电镜（TEM）证实，CyHV-3-QY08在KCF-1细胞中连续增殖。感染CyHV-3-QY08的KCF-1细胞产生了典型的细胞病变，其特征是严重的空泡化、细胞核变形和核染色质边缘化，这与以往的报道一致。纯化CyHV-3-QY08，并进行SDS-PAGE和TEM分析。结果表明，纯化的病毒粒子具有两种形态，由30多种明显的病毒多肽组成。感染实验表明，CyHV-3-QY08可使感染的锦鲤死亡率达到100%。基于CyHV-3-I/U的基因组序列，克隆了CyHV-3(I/U)-ORF136在CyHV-3- qy08中的同源序列并进行了测序。对CyHV-3-I/U-ORF136同源物的多次序列比对表明，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的分子标记，可用于区分亚洲分离株和欧美株。(C) 2011 Elsevier B.V.版权所有
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. (C) 2011 Elsevier B.V. All rights reserved.