Human 293T cell genomic DNA was prepared from terminal cocultures and 50 ng was used for high fidelity, PERV pol genespecific PCR reactions (Phusion polymerase; Finnzymes). 193 bp products were cloned using the Zero Blunt TOPO PCR Cloning kit (Invitrogen) and sequenced (University of Minnesota Advanced Genetic Analysis Facility). Sequence comparisons were performed using Sequencher software (Gene Codes Corp.) and publicly available Clustal W alignment algorithms (http://align.genome. jp/). Figure S1 Quantitative Real-time PCR Analyses. (A) Standard curves depicting Q-PCR data obtained using dilutions of a linearized PERV pol gene plasmid alone (squares) or diluted plasmid plus 10 ng of 293T cell genomic DNA (diamonds). Under both conditions, all template amounts (10 to 10 7 copies) amplified efficiently (the log-linear slope equations are shown). The correlation co-efficiency value (R 2 ), which reports the technical accuracy of the assay, is also indicated. The standard curve data points were the average of 2 independent reactions with deviations smaller than the symbols (i.e., C T errors for each point ranged from 0 to 0.4). (B) Two representative control Q-PCR datasets showing the amplification of PERV pol gene DNA from pig PK-15 cell genomic DNA (circles). Two additional control Q-PCR datasets showing that the PERV-specific primers fail to amplify product from uninfected human 293T cell genomic DNA (squares). The reaction threshold, 10 times the mean standard deviation of the background fluorescence level (BioRad), is indicated. (C) Representative co-culture Q-PCR amplification curves of PERV pol gene DNA. Template genomic DNA isolated from human 293T cells co-cultured with vector expressing PK-15 cells (diamonds) or human APOBEC3G-expressing PK-15 cells (triangles) was used. (D) Representative Q-PCR amplification curves of the 293T cell beta-actin gene, which served as an internal standard for quantifying the real-time PCR data. Raw Q-PCR data will be made available on request. Found at: doi:10.1371/journal.pone.0000893.s001 (9.93 MB TIF) Figure S2 APOBEC3G inhibits PERV transmission. (A) A graph showing the accumulation of PERV pol gene-specific PCR products in 293T cells co-cultured with a control cell line (V3) but not with an APOBEC3G-expressing cell line (G1). The data points were an average of two Q-PCR runs and the difference between each run was smaller than the plotted symbol. The experimental parameters were identical to those used in the experiments shown in Figures 1B and 2B. (B) Relative levels of reverse transcriptase(RT)-activity detected in soluble extracts of day 28 co-cultured 293T cells, which were used to generate the Q-PCR data shown in Figure S2A . Uninfected 293T cell lysates had a relatively high endogenous RT activity. Therefore, to help with the presentation of these data, this level was normalized to one and all of the other data were calculated relative to this value. The level of RT activity in PK-15 extracts was much higher than that of 293T cell extracts (+/2PERV) and it had reached saturation (out of range) when these data were collected. Found at: doi:10.1371/journal.pone.0000893.s002 (4.76 MB TIF) Figure S3 Pig APOBEC3F Is Expressed in PK-15 Cells and its Over-expression Does Not Markedly Inhibit PERV Transmission. (A) An image of an ethidium bromide-stained agarose gel showing the results of an RT-PCR amplification experiment using PK-15 cellular RNA and appropriate controls. The top panel shows that PK-15 and representative PK-15 derived clones all expressed pig APOBEC3F, as indicated by the specific 175 bp pig APOBEC3F PCR product (confirmed by DNA sequencing). 293T cell mRNA and a diluted pig APOBEC3F expression plasmid were used as negative and positive controls, respectively. A larger, non-specific band was apparent only in the 293T cell RT-PCR reactions. The bottom panel shows that a conserved, 236 bp beta-actin gene fragment could be amplified from both PK-15 cells and human 293T cells (but not from diluted plasmid DNA). Note that this primer set differs from the human-specific set used in the Q-PCR experiments. The sizes of the marker (M) DNA bands are shown. (B) An image of an ethidium bromide-stained agarose gel showing expression of plasmid-derived pig APOBEC3F in PK-15 cells after 26 days of continuous co-culture. Non-transfected (NT) cells and diluted APOBEC3F plasmid DNA (pDNA) provided negative and positive controls, respectively. The larger 319 bp (far right lane only) and smaller 190 bp bands are the specific PCR products of the first and second rounds of semi-nested PCR, respectively (confirmed by DNA sequencing). (C) A histogram summarizing the level of PERV transmission that was observed after 23 days of co-culturing human 293T cells with PK-15 cells expressing a vector control or over-expressing pig APOBEC3F. Two datasets, each with an independent PK-15 clone in three replica co-culture wells, were collected in parallel and averaged for each histogram bar. One standard error of the mean is shown. The experimental parameters are identical to those used in Figure 1B . Found at: doi:10.1371/journal.pone.0000893.s003 (8.52 MB TIF) Figure S4 Genetic Variation in Zoonosed PERV pol Gene Sequences. (A) Sequences of the PERV pol gene fragments cloned from 293T cells co-cultured with control vector-expressing PK-15 cells. The number of times that each sequence was recovered is shown (N). Experiments 1 and 2 used genomic DNA prepared from the 293T cells used to generate the data shown in Online Figure S2 (day 28 samples) and Figure 2B (day 23), respectively. The most frequently detected 147 bp PERV pol gene sequence is shown in its entirety (which together with PCR primers makes up the 193 bp product shown in Figure 5 ). Identical nucleotides in other sequences are represented by dashes and non-identical nucleotides by the indicated DNA bases. GenBank accession numbers are shown for pol gene fragments with 100% identity to previously reported sequences. (B) Sequences of the PERV pol gene fragments cloned from 293T cells co-cultured with control APOBEC3G-expressing PK-15 cells. Parameters are identical to those described above. Found at: doi:10.1371/journal.pone.0000893.s004 (0.05 MB DOC) Experimental infection of H5N1 HPAI in BALB/c mice BACKGROUND: In 2005 huge epizooty of H5N1 HPAI occurred in Russia. It had been clear that territory of Russia becoming endemic for H5N1 HPAI. In 2006 several outbreaks have occurred. To develop new vaccines and antiviral therapies, animal models had to be investigated. We choose highly pathogenic strain for these studies. RESULTS: A/duck/Tuva/01/06 belongs to Quinghai-like group viruses. Molecular markers – cleavage site, K627 in PB2 characterize this virus as highly pathogenic. This data was confirmed by direct pathogenic tests: IVPI = 3.0, MLD(50 )= 1,4Log10EID(50). Also molecular analysis showed sensivity of the virus to adamantanes and neuraminidase inhibitors. Serological analysis showed wide cross-reactivity of this virus with sera produced to H5N1 HPAI viruses isolated earlier in South-East Asia. Mean time to death of infected animals was 8,19+/-0,18 days. First time acute delayed hemorrhagic syndrome was observed in mice lethal model. Hypercytokinemia was determined by elevated sera levels of IFN-gamma, IL-6, IL-10. CONCLUSION: Assuming all obtained data we can conclude that basic model parameters were characterized and virus A/duck/Tuva/01/06 can be used to evaluate anti-influenza vaccines and therapeutics. Influenza A (H5N1) virus now becomes a real threat for humans. Since 1997, when first human case of H5N1 HPAI had been reported, more than 317 people were infected and 191 died [1] . Before 2005 attention was attracted to Thailand, Vietnamese and Indonesian viruses. In the beginning of 2005 outbreak on Quinghai lake occurred [2] . Later "Quinghai-like" viruses spreaded to most part of Russia, European countries and Africa and caused numerous outbreaks. Only in Russia more than 1 million of different species and sorts of poultry died and been slaughtered [3] . Confirmed cases in Azerbaijan, Egypt, Iraq, and Turkey was caused by Quinghai-like viruses. Earlier HPAI viruses were investigated in mice [4, 5] and murine models were successively used for reverse genetics made influenza vaccines [6] . It was shown that H5N1 HPAI viruses could have different pathogenicity for mice [7] . Several molecular markers were choused to explain differences. Multibasic cleavage site with 627K in PB2 designate to highly pathogenic phenotype for mice. Also important role of pulmonary cytokines elevation was highlighted [8] . Combination of adaptation for wild waterfowl and high virulence for mammals makes Quinghai-like viruses presumably pandemic. Also, in future, because of ability for rapid spreading for long distances, this group of viruses can appear in North and South America and cause outbreaks.