Megalocytivirus infection is a major threat in rock bream aquaculture in Korea. To produce a highly concentrated megalocytivirus, primary cells, established cell line and persistently infected cell line were used in this study. Megalocytivirus was inoculated in primary fin cell cultures of red sea bream (Pagrus major), rock bream (Oplegnathus fasciatus), olive flounder (Paralichthys olivaceus) and black sea bream (Acanthopagrus schlegelii) and produced at similar concentrations of 10^{8.99 − 9.88} viral particles/mL in all cultures while produced 10^7.31 viral particles/mL in grunt fin (GF) cell line. Since only red sea bream fin culture was amenable to subculturing for more than 100 times, it was established into Pagrus major fin (PMF) cell line. A persistently infected PMF cell line (PI-PMF) was obtained by continuous subculturing every 7 days as a batch culture system (PI-PMF-B) after infecting with megalocytivirus. Virus in supernatant of PI-PMF-B was maintained at high concentrations throughout over 50 consecutive subcultures in a relatively narrow range from 10^8.33 to 10^8.94 viral particles/mL with high level of CPE. For a more efficient and convenient production, a semi-batch culture system (PI-PMF-S) was developed in which culture media were exchanged at intervals of 3 days without subculturing for more than 50 media exchanges. Despite low virus productivity in a single cell (specific virus productivity, SVP), total cell number was increased in PI-PMF-S, allowing us to efficiently obtain a much higher concentration of virus (10^8.56 to 10^9.75 viral particles/mL) than in PMF-B. This is the first study to report detailed new methods for continuous and efficient production of high concentrations of megalocytivivrus with characterization of viral propagation in persistently infected cells.

在韩国，鲷鱼感染是鲷鱼养殖的主要威胁。为了产生高度浓缩的巨轮病毒，本研究使用了原代细胞，建立的细胞系和持续感染的细胞系。巨轮病毒被接种在红鲷（Pagrus major），石鲷（Oplegnathus fasciatus），比目鱼（Paralichthys olivaceus）和黑鲷（Acanthopagrus schlegelii）的原鳍细胞培养物中，并产生相似浓度的10 ^8.99-9.88病毒颗粒/ mL在所有培养物中均产生10 ^7.31鳍（GF）细胞系中的病毒颗粒/ mL。由于仅红鲷鳍养殖适合于传代超过100次，因此它被建立到Pagrus主要鳍（PMF）细胞系中。巨噬病毒感染后，以分批培养系统（PI-PMF-B）的方式每7天连续继代培养，从而获得了持续感染的PMF细胞系（PI-PMF）。PI-PMF-B上清液中的病毒在连续50多次传代培养中均保持在高浓度，范围从10 ^8.33到10 ^8.94相对较窄高水平CPE的病毒颗粒/ mL。为了提高生产效率和便利性，开发了一种半分批培养系统（PI-PMF-S），该系统每隔3天交换一次培养基，而无需再进行50次以上的培养基交换。尽管单个细胞中的病毒生产率较低（特定病毒生产率，SVP），但PI-PMF-S中的总细胞数却增加了，这使我们能够有效地获得更高浓度的病毒（10 ^8.56至10 ^9.75病毒颗粒/ mL）比PMF-B 这是第一项报道详细详细的新方法的研究，该新方法可连续有效地生产高浓度的巨轮生病毒，并表征持续感染细胞中的病毒繁殖。