The method given below for purifying honey bee viruses is simple but only suitable for non-enveloped viruses, which fortunately covers the vast majority of RNA viruses. However, this method is unsuitable for viruses containing membranes, such as Apis mellifera filamentous virus. There are a number of enveloped virus families that have insect-infecting members and there may be more enveloped honey bee viruses to be discovered. The various buffers, solvents and centrifugation conditions for individual viruses are given in Table 4. Most of the buffers shown are the phosphate buffers developed by Bailey and Ball (1991), between 0.01 and 0.5M and of neutral pH (between 7.0 and 8.0). In most cases, TRIS.Cl buffers of similar molarity and pH will perform equally well. Similarly, chloroform can be substituted for the more toxic ether/carbon tetrachloride combination for extract clarification. Nonionic detergents such as Triton X100 (0.05%), BRIJ-58 (2%) and/or sodium deoxycholate (0.2%) are also common agents for lipid solubilisation and extract clarification during virus purification. 0.1 M ascorbic acid is a common alternative to DIECA as antioxidant. Conduct as much of the purification as possible at 4oC (on ice). With each purification step, there is a considerable loss of yield, particularly during the high-speed and gradient centrifugation steps. As much as 80% of the primary extract can be lost during purification. It is therefore important to consider how pure the virus preparation needs to be for your experiments. For infection experiments, purity may be less important while for developing a specific antiserum, high purity is essential. The high-speed and gradient centrifugation steps are excellent for separating the virus from other cellular contents and particles, but are not suitable for separating different virus species: they all have very similar densities.