5.2. Starting material

Often the starting material for propagation is a previous virus preparation that has been checked for the absence of contaminating viruses and retained as a pure isolate. Virus preparations can, however, lose infectivity during prolonged storage. For example deformed wing virus (DWV) and its relatives kakugo virus (KV) and Varroa destructor virus-1 (VDV-1), are particularly sensitive to decay during storage. Since neither serological nor molecular assays can distinguish between degraded or intact virus particles, they are not reliable methods for determining the infectivity of a preparation. Furthermore, such well-characterized and precious reference material is often in limited supply and highly valuable as a historical “reference” isolate for future experiments. Such reference material can be stored long-term either as freeze-dried bees/pupae/larvae, or as a (semi)purified virus solution in an appropriate virus purification buffer (Table 4) and stabilized by 50% glycerol. It is therefore advisable, particularly for infectivity tests but also for propagation, to first prepare a “working” inoculum, by injecting or feeding a small number of bees (larvae, pupae or adults; around 5-10 individuals) with a small amount of the pure reference material. This also serves as an infectivity test for the viability of the stored reference material. After incubating for the appropriate time, a crude extract should be prepared from the bees, the purity and virus concentration of this extract determined, and then this working extract should be used for large-scale propagation or infectivity tests within the next few of weeks.