[Interaction mechanism of marine birnavirus (MABV) in fish cell lines]

Marine birnavirus (MABV) is a member of the genus Aquabirnavirus of the family Birnaviridae. MABV is an unenveloped icosahedral virus about 60 nm in diameter with two genomes of double-stranded RNA. MABV adsorbed not only onto the cell surfaces of susceptible (CHSE-214 and RSBK-2) cells but also onto resistant (FHM and EPC) cells. Furthermore, the virus entered into the cytoplasm through the endocytotic pathway in CHSE-214, RSBK-2 and FHM cells but did not penetrate EPC cells. The virus was found to bind to an around 250 kDa protein on CHSE-214, RSBK-2, FHM and EPC cells. The syntheses of viral proteins pVP2, NS and VP3 and further proteolytic processing after viral infection were examined by using Western blot analysis. pVP2, NS and VP3 were detected in the cytosolic fractions of CHSE-214, RSBK-2 and FHM cells at 4 h after infection. At this time, VP3 underwent further proteolytic processing in the cytosolic fractions of CHSE-214 and RSBK-2 cells. The expression of pVP2, NS and VP3 increased and pVP2 and NS also underwent further proteolytic processing similar to VP3 in the cytosolic fractions of CHSE-214, RSBK-2 and FHM cells at 8 h after infection. The further proteolytic processing of VP3 was detected in the nuclear fractions of CHSE-214, RSBK-2, but VP3 was detected as a single band in the nuclear fraction of FHM cells. pVP2 and NS were detected as thin bands only in the nuclear fractions of CHSE-214 cells. The results of Western blot analysis demonstrated that pVP2, NS and VP3 are localized in the nuclear fraction when they were independently expressed in CHSE-214, RSBK-2, FHM and EPC cells. The expression pattern in the cytosolic fraction was identical among the four cell lines when pVP2 and NS were independently expressed. However, pVP2 and NS were not detected in the nuclear fraction of CHSE-214 cells. Further proteolytic processing of VP3 was detected in both cytosolic and nuclear fractions of RSBK-2 ,FHM and EPC cells (Low level in EPC cell), but not in CHSE-214 cells when VP3 was independently expressed. Then, the processes of preVP2 to form morphological assemblages in the presence of VP3 or the cleavage of VP3 into two proteins in CHSE-214 cells were studied. When preVP2- and VP3 were co-expressed, virion like particles (64 nm, diameter) were observed close to the nuclear membrane by electron microscopy. The co-expression of preVP2 and the cleaved VP3 proteins led to an efficient assembly of tubules (22 nm, diameter). Further important finds will be obtained by this infection system using 4 fish cell lines in the next couple of years.