To analyze the H5N1 viral load and quantify proinflammatory factors in different tissues, real-time RT-PCR was performed with a Strategene detection system using a fluorescently labeled TaqMan probe to enable continuous monitoring of amplicon formation. The primer and probe of H5 HA gene is from the WHO-released primer sets [50] . The primers and probes of proinflammatory factors and beta-actin gene were obtained from the literature [51, 52] . The concentration of primer and probe used was 40 mM and 10 mM, respectively. The reaction was completed in a total volume of 25 ml performed by QuantiTect Probe PCR Kit (Qiagen, Germany). The reaction mixture was incubated with 5 ml DNase-treated total RNA (the template which was used to amplify b-actin gene was performed by 100-fold dilution) at the following temperature cycles. First, the reverse transcription reaction was completed by 1 cycle at 50uCfor 30 min. Next, H5N1 HA gene, cytokine/chemokine gene and house keeping (bactin) genes were amplified by 1 cycle at 94uC for 15 min and 45 cycles at 94uC for 15 s, 55uC for 30 s, and 72uC for 30 s each. As described in previous report [53] , the standard curve was generated using serial dilution of in vitro transcribed standard RNA (from ,10 to 10 7 copies). The viral load and cytokines/ chemokine levels are presented as the log 10 value of the ratio between copies of the target gene and b-actin gene.