PMF cell persistently infected with Megalocytivirus (PI-PMF) has been developed to yield a high concentration of virus. For characterization of PI-PMF, we asked, what proportion of cells are producing viruses, is there an apoptosis even in persistency, and what biological changes were induced in PI-PMF by persistent infection in terms of immune responses, virulence and genetic modification in the genomic region of repeating sequences?
With limiting dilution method seeding a single cell in each well of 96 well plate, it was confirmed that more than 13.85% cells in PI-PMF are not producing virus. From virus negative wells, we isolated four virus-free cell clones (VF cells) by repeated limiting dilution. With inoculation of Megalocytivirus, all those four cloned cells were re-infected. Although slightly lower virus production was observed in supernatant of each VF cell after reinfection of Megalocytivirus compared to that of PI-PMF, those results allowed us to assume that VF cells are not separated group but present with infected cells to establish an equilibrium between host cell maintenance and virus production.
In characterization of PI-PMF cells changed by persistency, we found no apoptotic DNA fragmentation in total extracted DNA and allowed us to presume that the absence of apoptosis could be related to persistency. Additionally, we found the increased level of Mx gene expression, over 20-fold than that of PMF, as reported in BB (Barramundi Brain) cells persistently infected with VNN (Virus Nervous Necrosis virus). Although it should be studied further the meaning of sustained innate immune responses, both no apoptosis and increased immune response are sure to be elements necessary to exist as a persistent infection state in PI-PMF cells.
Finally, virus obtained from PI-PMF (VP) showed higher genetic polymorphism in ORF25 region carrying repeated sequences compared to that appeared in the genome of wild type strain (V0) obtained from infected rock bream (Oplegnathus faciatus) in filed. However, similar pathogenicity of these two virus isolates to rock bream suggested that the repeated passage and the genetic change in repeating sequences were not directly responsible for the virulence of Megalocytivirus.