Over-passage of epithelioma papulosum cyprini (EPC) cells increased viral hemorrhagic septicemia virus (VHSV) replication

Abstract

Vaccines based on inactivated or attenuated viruses can be a way to prevent viral hemorrhagic septicemia virus (VHSV) disease, and the efficiency of viral production is a critical factor that can determine the practical use of developed vaccines in aquaculture farms. To know the effects of epithelioma papulosum cyprini (EPC) cells over-subculture on VHSV replication, the VHSV titer produced from high-passage EPC cells (subcultured more than 200 times in our laboratory) was compared to the titer produced from low-passage EPC cells (subcultured 5–15 times). Furthermore, to know whether immune factors are involved in VHSV titers, differences not only in the expression of Mx1 and ISG15 genes but also in the apoptosis progression by VHSV infection between high- and low-passage EPC cells were analyzed. The VHSV titers from high-passage EPC cells were significantly higher than titers from low-passage EPC cells, suggesting that the changed properties of EPC cells by over-subculture were favorable for VHSV proliferation. The DNA laddering of high-passage EPC cells by VHSV infection took a longer time than that of low-passage EPC cells, suggesting that over-subculture might delay apoptosis in VHSV infected EPC cells, and the delay of apoptosis by over-subculture can be thought as one of the factors that increased VHSV titers in high-passage EPC cells. The increased folds of Mx1 and ISG15 genes in high-passage EPC cells were significantly lower than those in low-passage EPC cells when exposed to either poly (I:C) or VHSV. However, the expression levels of Mx1 and ISG15 genes of high-passage EPC cells that were not stimulated with poly I:C or VHSV were almost equal to or higher than the expression levels of low-passage EPC cells that were exposed to poly (I:C) or VHSV. This result suggests that high-passage EPC cells were already in an excited state in type I interferon responses without any stimulants. The full open reading frame (ORF) sequences of Mx1 gene between high- and low-passage EPC cells were completely same. However, there were some differences in the amino acids sequences of ISG15 gene between high- and low-passage EPC cells, suggesting that ISG15-mediated pathways might be different between high- and low-passage EPC cells, which might influence on the replication of VHSV. The present results showed that the changed properties of EPC cells by over-subculture were favorable for VHSV proliferation.