Cryopreservation of a Virus (HaV) Infecting a Harmful Bloom Causing Microalga, Heterosigma akashiwo (Raphidophyceae)

Establishment of a technique for the stable preservation of isolated viruses is highly desirable for use in studies on microalgal viruses. In addition, successful preservation will ensure minimal genetic change in the virus culture, a phenomenon which may occur during the repeated trans fer to a fresh host culture. However, storage techniques for algal viruses have not been intensively discussed so far. HaV is a virus infecting the harmful bloom causing alga, Heterosigma akashiwo (Raphidophyceae). ') Although the optimum temperature range for HaV proliferation is 2025•Ž, at this temperature range loss of infectivity occurs. 2) Thus, to maintain HaV infectivity, frequent transfer of HaV lysate to an actively growing host culture is neces sary. However, the operation not only requires much time and labor but is also accompanied with the risk of unex pected genetic change of the virus culture, contamination by bacteria and mislabelling of virus strains. In addition, establishment of a stable preservation technique for HaV is needed from the viewpoint of application of HaV as a microbiological agent to control H. akashiwo red tides. ') The objective of the present study is to establish the opti mum condition for the cryopreservation of HaV. An axenic clone of HaV (strain HaV01) and a clonal and axenic strain of H. akashiwo (strain NM96) were used in this study.4) Incubation conditions are as shown previously.1) An exponentially growing culture of H. akashiwo NM96 was inoculated with HaV01 and incubated for three to four days. The resultant lysate was titrated by the extinc tion dilution method.1) To examine the effect of cryoprotectants for HaV, the viral lysate was mixed with an equal volume of SWM3, 20% glycerol in SWM3, 20% dimethyl sulfoxide (DMSO) in SWM3 and 20% sucrose in SWM3, respectively. Aliquots of each mixture (0.5 ml) were poured into a serum tube (SUMILON MS-4501B), and stored at -20•Ž, -80•Ž and -196•Ž (liquid nitro gen), respectively. After two weeks of storage at each tem perature, the samples were thawed by placing the tubes under flowing tap water until a particle of frozen liquid ap proximating the size of a grain of rice remained, then the solution was vigorously stirred until completely thawed. Virus titer was then determined by the extinction dilution method.1) For the DMSO solutions, the cryoprotective effect at -196•Ž was re-examined setting the final concen tration at 0, 5, 10, 20 and 30%, and a storage period of two weeks. The virus titer of a sample added with 10% DMSO and stored at -196•Ž for 11 months was also esti mated. The effect of temperature and cryoprotectants on the preservation of HaV is shown in Fig. 1. Without the addi tion of any cryoprotectants, no viral titer was detected af ter two weeks of storage at all of the temperatures tested. Furthermore, it was confirmed that all of the cryoprotectants tested were ineffective for preservation at -20•Ž and -80•Ž . In contrast, 0.01% and 2.9% of the initial titer were maintained after two weeks storage at -196•Ž when 10% glycerol and 10% DMSO were added