The total RNA of infected VeroE6 cells or mouse liver, spleen, kidneys and brain were extracted with Trizol reagent (Invitrogen). First-stranded cDNA was synthesized with a random hexamer by RTG YouPrime RXN Beads (GE Healthcare, Bucks, UK) according to the manufacturer's instructions. PCR primers which anneal to nucleotide 411 to nucleotide 430 (M430F: 59-ATG GCA GGG ATT GCA ATG AC-39) or nt.1041 to 1060 (M1041R: 59-ACT GCA AAG GGC ACA ACC TC-39) of anti-viral-sense M were used for PCR reaction. PCR was performed for 30 cycles at 94uC for 40 sec, 55uC for 1 min, and 72uC for 1 min using the Expand High Fidelity PCR System (Roche, Mannheim, Germany). The PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Germantown, MD), digested with Sac I and then separated on a 1% agarose gel.