Spring Viraemia Of Carp Virus Glycoprotein Expressed In Insect Cells And Transduction Of Fish Cells By Recombinant Baculovirus

Spring viraemia of carp virus (SVCV) is a member of the genus Vesiculovirus of the family Rhabdoviridae. Spring viraemia of carp (SVC), which is caused by SVCV is an acute haemorrhagic and contagious viraemia disease in common carp. The glycoprotein (G) of Spring viraemia of carp virus (SVCV/G) is a major antigen, which plays an important role in determining the serological property of the SVCV, and is the target protein for the development of vaccines and diagnostic reagents. In this study, a recombinant transfer plasmid pFastBac-SVCV/G which had been constructed in our laboratory was transformed into competent DH10Bac, which contained baculovirus shutter vector (bacmid) and helper vector. The recombinant bacmid (rBacmid-SVCV/G) was obtained from transformed DH10Bac, and was identified by blue/white selection and PCR analysis. Then the rBacmid-SVCV/G was transfected into sf9 cells with Cellfectin Reagent and the recombinant baculovirus Bac-SVCV/G was obtained in sf9 cells. Bac-SVCV/G can induce insect cell fusion, as observed by invert microscopy. SVCV/G was detected on the surface of the the insect cells by indirect immunofluorescence assay. In previous studies, it has been found the glycoprotein (G) of vesicular stomatitis virus (VSV/G) can also expressed on the surface of insect cells, and mediate insect cell fusion. For comparison of the cell fusions mediated by SVCV/G and VSV/G, and convenience for the determination of baculovirus titer, three recombinant baculoviruses were constructed, and named Bac-SVCV/G-CMV-EGFP, Bac-VSV/G-CMV-EGFP, Bac-CMV-EGFP. The SVCV/G expressed in insect cells was detected by indirect immunofluorescence assay and Western blot. Through comparison of the cell fusions mediated by SVCV/G and VSV/G, it was found that SVCV/G can cause more severely insect cell fusions.Studies on transduction of mammalian cells have shown that baculovirus can be used as an effective vector for gene delivery. However, previous studies have found the gene delivery efficiency to be very low in differentiated fish cells. For the purpose of enhancing the transduction efficiency of baculovirus in fish cells, in this study, the recombinant baculovirus Bac-CMV-EGFP containing cytomegalovirus immediate-early (CMV-IE) promoter and enhanced green fluorescent protein (EGFP) gene as the reporter gene was employed to reexamine the transduction efficiency of baculovirus in fish cells. The transduction efficiency of recombinant baculovirus was measured by flow cytometry and the persistence of EGFP was monitored by inverted fluorescence microscope. Our findings sharply contrast previous studies. Firstly, we demonstrated that baculovirus can mediate highly efficient gene delivery into differentiated fish cells. The transduction efficiency of baculovirus was nearly 100% in epithelioma papulosum cyprini (EPC) cells and fathead minnow muscle (FHM) cells, followed by chinook salmon embryo (CHSE-214), ctenopharyngodon idellus kidney (CIK), Channel catfish ovary (CCO). Rainbow trout gonad (RTG-2) cells can be transduced, although the transduction efficiency was not high. Furthermore, reporter gene expression in transduced EPC cells could persist for at least 80 days, which indicated transgene expression can persist for a lengthy period of time in fish cells. Finally, we found that growth medium is superior to PBS as the surrounding solution to enhance the transduction efficiency in some fish cells.