293 cells in 6-well plates were transfected with 2 mg of in vitrosynthesized RNA transcripts encoding rLuc or MP-12 NSs by TransIT mRNA transfection kit (Mirus). Cells were mock-treated or treated with ActD (5 mg/ml) immediately after RNA transfection. Total RNA were extracted by using an RNeasy Mini kit (Qiagen) at 16 h post-transfection. For each sample, we used 500 ng of RNA to synthesize 1 st strand cDNA by High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). Real-Time PCR was performed at the Real-Time PCR core facility, Sealy Center for Cancer Cell Biology, UTMB. We used an Applied Biosystems made-to-order 206 assay mix of primers and TaqMan MGB probes (FAM dye-labled) for our target gene PKR (Applied Biosystems: assay ID#: Hs01091592_m1) and predeveloped an 18S rRNA (FAM-dye labelled probe) TaqMan assay reagent (Applied Biosystems: 4352930E) for endogenous control. Separate tubes (singleplex) real-time PCR was performed with 40 ng cDNA for both target gene and endogenous control by using a Taqman Gene expression master mix (Applied Biosystems: 4370074). The cycling parameters for real-time PCR were: UNG activation at 50uC for 2 min, AmpliTaq activation at 95uC for 10 min, denaturation at 95uC for 15 sec, and annealing/extension at 60uC for 1 min (repeat 40 times) on ABI7000. Duplicate C T values were analyzed by the comparative C T (DD C T ) method, as described by the manufacturer (Applied Biosystems). The amount of target (2 2DDCT ) was obtained by normalized to endogenous reference (18S rRNA) and relative to a calibrator (one of the experimental samples). Figure S1 Effects of ActD treatment on host translation activities. (A) 293 cells cultured in 10-cm dishes were treated with 5 mg/ml of ActD (ActD) or were left untreated (Mock). Cells were harvested at 16 h post-ActD-treatment in 900 ml of Lysis buffer (50 mM Tris-HCl, pH 7.5; 5 mM MgCl2; 100 mM KCl; 1% Triton X-100; 100 mg/ml cycloheximide; and 0.5 mg/ml heparin) on ice for 5 min. The cytoplasmic lysates were collected after the removal of nucleus by centrifugation at 10,0006g for 5 min. The lysates were loaded onto a 10-50% linear sucrose gradient containing 50 mM Tris-HCl, pH 7.5; 5 mM MgCl2; 100 mM KCl; 0.5 mM dithiothreitol; 100 mg/ml cycloheximide; and 0.5 mg/ml heparin, and centrifuged at 38,000 rpm for 3 h at 4uC using a Beckman SW41 rotor. The gradients were pumped by syringe pump (Brandel) and analyzed by a density gradient fractionator (Brandel) connected to an ISCO UA-6 (ISCO Inc.) at the absorbance of 254 nm according to the manufacturer's instructions. The data were representative of two independent experiments. (B) 293 cells were transfected with in vitrosynthesized rLuc RNA transcripts and mock-treated (no drug) or immediately treated with 5 mg/ml of ActD (ActD) or 50 mg/ml of a-amanitin (Amanitin). Luciferase activities were measured at 16 h post-transfection. The data shown in the graphs (mean+/ 2standard deviation) were obtained from three independent experiments with p values by using Student's t-test (*: p,0.05). Found at: doi:10.1371/journal.ppat.1000287.s001 (4.02 MB TIF) Figure S2 Effects of different concentrations of ActD or aamanitin on eIF2a phosphorylation and N protein accumulation in rMP-12-infected cells. VeroE6 cells were infected with rMP12-rLuc at an moi of 3, and then treated with different concentrations of ActD or a-amanitin. Cell extracts and culture supernatants were harvested at 16 h.p.i. Note that ActD suppresses about 80% of RNA polymerase I activity at 0.04 mg/ml, and about 80% of RNA polymerase II and III at 4.0 mg/ml [28] , while a-amanitin suppresses nearly 100% of RNA polymerase II and about 50% of RNA polymerase III at 50 mg/ml [64] . (A) Western blot analysis of N protein, phosphorylated eIF2a, total eIF2a and a-actin in each cell extract. (B) Top panels represent the relative abundance of phosphorylated eIF2a and total eIF2a. The relative abundance of phosphorylated eIF2a and total eIF2a in the untreated cells represents 100%. The middle panels and the bottom panels represent the abundance of N protein and the virus titers, respectively. The results were obtained from three independent experiments with p values by using Student's t-test (*: p,0.05). Found at: doi:10.1371/journal.ppat.1000287.s002 (4.33 MB TIF) Figure S3 Effects of pan-caspase inhibitor Z-VADfmk on PKRmediated eIF2a phosphorylation in infected cells. VeroE6 cells were mock-infected (Mock) infected with rMP12-rLuc (rMP12-rLuc) and then treated with 5 mg/ml ActD (Act) or 50 mg/ml of aamanitin (Ama) or mock-treated (M) in the presence or absence of 100 mM of Z-VADfmk. Cells and culture supernatants were harvested at 16 h.p.i. (A) Western blot analysis of eIF2a, phosphorylated eIF2a, cleaved caspase 3 using anti-Cleaved Caspase 3, Asp175, antibody (Cell Signaling Tech. #9661), and a-actin in the absence (Top) and presence (Bottom) of Z-VADfmk. The data are representative of three independent experiments. (B) The relative abundance of phosphorylated eIF2a and total eIF2a. The relative abundance of phosphorylated eIF2a and total eIF2a in the mock-infected, mock-treated cells represents 100%. The average and standard deviation from three independent experiments were shown (*: p,0.05 compared to mock-treated cells). (C) Virus titers of rMP12-rLuc from three independent experiments were shown (*: p,0.05 compared to mock-treated cells). Found at: doi:10.1371/journal.ppat.1000287.s003 (2.29 MB TIF) Influenza Virus (H5N1) in Live Bird Markets and Food Markets, Thailand A surveillance program for influenza A viruses (H5N1) was conducted in live bird and food markets in central Thailand during July 2006–August 2007. Twelve subtype H5N1 viruses were isolated. The subtype H5N1 viruses circulating in the markets were genetically related to those that circulated in Thailand during 2004–2005. Infl uenza virus (H5N1) was identifi ed in 12 of 930 samples tested. In November 2006, a total of 5 samples with infl uenza virus (H5N1) were isolated from 1 healthy chicken and 4 visceral organs obtained from 1 live bird market (chicken) and 3 different food markets (moor hen, water cock, and quail). In December 2006, a total of 5 samples with infl uenza virus (H5N1) were isolated from 5 visceral organs (quail, water cock) from 1 food market. In January 2007, a total of 2 samples with infl uenza virus (H5N1) were isolated from 2 healthy ducks obtained from 1 live bird market. In the study, 7 isolates were sequenced for whole genome analysis, and the remaining 5 To analyze the isolates, nucleotide sequences were compared with those of infl uenza subtype H5N1 viruses in Thailand, People's Republic of China, Vietnam, Indonesia, Lao, Myanmar, and Cambodia. The sequences were aligned by using the DNASTAR program (3) to elucidate and compare the genetic changes. Phylogenetic analysis was conducted by applying the PAUP program (4) with the neighbor-joining algorithm and using branch swapping and bootstrap analysis with 1,000 replicates.