5.5.1. Virus infection

The most common method of infecting tissue culture cells is through passive co-incubation of purified virus particles with the cells, allowing the natural processes of virus entry to establish an infection (Minor, 1985; Gantzer et al., 1998; Rhodes et al., 2011; Amdiouni et al., 2012). However, the virus particles (or the naked viral RNA genome) can also be forced into the cells using electroporation, which involves a short high-voltage pulse of electricity to temporarily open up the cell membrane to allow foreign elements to enter the cell, or chemical-mediated transfection, where a combination of membrane-active ions and concentrating agents interact to encourage the uptake of the virus or nucleic acid into the cell (e.g. Boyer and Haenni, 1994; Benjeddou et al., 2002; Ongus et al., 2006; Yunus et al., 2010).

Whichever virus transfection protocol is chosen, it is essential that the virus preparation is free of bacteria or fungi to prevent contamination of the tissue culture (Minor, 1985; Gantzer et al., 1998). Bacteria, fungi and their spores can be effectively removed from a virus preparation using microfilters with appropriate pore size, depending on the size of the virus (Gantzer et al., 1998; Rhodes et al., 2011; Amdiouni et al., 2012), with 0.2 µm suitable for purifying most small, enteric picorna-like viruses of around 30-60nm (Rhodes et al., 2011; Amdiouni et al., 2012), which includes most of the honey bee RNA viruses (Table 2).