Isolation and characterization of a novel strain (YH01) of Micropterus salmoides rhabdovirus and expression of its glycoprotein by the baculovirus expression system / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus (MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary (GCO) cell line and displayed virion particles with atypical bullet shape, 300-500 nm in length and 100-200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein (1-458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification. Based on polymerase chain reaction (PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.

Development and characterization of two new cell lines from common carp, Cyprinus carpio (Linn)

Two new cell lines (CCF and CCH) were established from fin and heart tissues of common carp, Cyprinus carpio. The cells were optimally maintained in Leibovitz-15 medium supplemented with 10 percent fetal bovine serum (FBS) and 10 ng/ml of basic fibroblastic growth factor (bFGF). The effects of temperature, concentration of FBS and bFGF on the growth of CCF and CCH cells were examined. The temperature ranged from 24 to 32 °C for good growth of the cells. The growth rate of cells was higher in medium containing 10 percent FBS and the addition of bFGF to the medium significantly increased the growth rate. The CCF cells were found to be epithelial, while the CCH cells were fibroblastic in nature. The cytogenetic analysis of the cell lines revealed a diploid number of 100 chromosomes in C. carpio. The viability of CCF and CCH cell lines were 70 and 72 percent, respectively, after six months of storage in liquid nitrogen (-196 ° C). Molecular characterization of the cell lines using 16S rRNA and Cytochrome Oxidase Subunit I (COI) revealed the origin of the cell lines. These new cell lines will be useful for isolation of fish viruses and other in vitro biotechnological studies.