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These mice were daily investigated and weighed for 14 days. Mice which lost more than 30% of their body weight were euthanized. MLD50 were calculated using the Reed-Muench method (Reed and Muench, 1938). Polymerase activity assay To detect the polymerase activity of viruses, the plasmid pMD18T-NP-LUCI was constructed. In a brief explanation, the luciferase gene flanked with the non-coding region of NP of DK212 was amplified, using pGL-3 control (Promega) as template, by primer L-NPFL-NPR. It was then cloned into pMD18-T. 293T cell was cultured in a 12-well plate overnight. When the monolayer formed approximate 90%, they were transfected with 0.4��g of pHH-NP, pHH-PA, pHH-PB1, and pHH-PB2, and 0.1��g of pMD-18T-NP-LUCI, Telomerase as well as 0.1��g pRL-SV40 (Promega) as an internal control. After transfection for 24h, cell lysate was prepared and the luciferase yield was tested by using the dual-luciferase reporter assay system (Promega), with a single tube luminometer (Promega). Relative activity of the polymerase was normalized to Renilla gene expression. The results were calculated as means �� standard deviations from three independent experiments, using SPASS (Version 11.5). Biosafety and animal handling The laboratory and mice experiments were carried out under BSL-3 conditions in compliance with biosafety committee of South China Agriculture University approved protocols. The handling of mice was performed in accordance with experimental animal administration and ethics committee of South China Agriculture University approved guideline. Results The virulence of DK212 was stronger find more than QL90 MDV3100 research buy to BABL/c mouse To compare the pathogenicity of DK212 and QL90, mice were intranasally infected with the corresponding virus at a dose of 106 embryo infective dose 50 (EID50). DK212 quickly caused 23.97% body weight loss and killed all mice by 7 DPI at a dose of 106 EID50. Virus was isolated from the brain, spleen, kidney, and lung, with a mean titer between 1.67 and 6.25 log10EID50 on 3 DPI. Furthermore, the virus titer of DK212 was higher than that of QL90 in the brain and kidney (p