Characterization of a New Cell Line from Caudal Fin of Koi, Cyprinus Carpio Koi, and First Isolation of Cyprinid Herpesvirus 3 in China

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.