For determination of FeLV proviral loads, total nucleic acids were extracted from a blood volume containing 10 6 white blood cells using the MagNa Pure LC Total Nucleid Acid Isolation Kit (Roche Diagnostics AG, Rotkreuz, Switzerland). The extracted total nucleic acids were analyzed by real-time TaqMan PCR as described in [39] using the 2· TaqMan Ò Fast Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on a ABI 7500 sequence detection system (Applied Biosystems) and under the following cycling conditions: an initial denaturation of 20 s at 95°C was followed by 45 cycles of 95°C for 3 s and 60°C for 30 s. For each run, a glyceraldehyde-3-phophate dehydrogenase (GAPDH) pseudogene of which one copy is present in the genomic DNA of feline cells [33] was also quantified as described [39] using the 2· TaqMan Ò Fast Universal PCR Master Mix and the same PCR run conditions as for FeLV provirus. FeLV proviral DNA amounts were normalized to feline GAPDH by dividing FeLV copy numbers by fGAPDH copy numbers to calculate FeLV copies per cell. Viral RNA in plasma samples was extracted from 200 lL of plasma (either from 5-sample pools or from single samples) using the MagNa Pure LC Total Nucleic Isolation Kit and quantified by real-time TaqMan reverse transcriptase (RT)-PCR as described [39] using a ABI 7500 sequence detection system.