Additional file 1: Table S1. of Development and utility of an in vitro, fluorescence-based assay for the discovery of novel compounds against dengue 2 viral protease

Primers used in the amplification of the target sequence. Lower case letters denote viral sequence; italicized letters denote sequence comprising the glycine linker region. Letters in bold indicate restriction endonuclease recognition sequences. Figure S1. Fraction collections using size exclusion chromatography. (A) Absorbance detected per each fraction as a function of time. (B) Coomassie SDS-PAGE analysis, (C) anti-histidine Western blot. M = marker, W = 20 mM, 1 = 100 mM, 2 = 200 mM, 3 = 400 mM, 4 = 800 mM Imidazole. Figure S2A. Comparison of different flaviviral protease substrates. Single concentration of substrate (1000 μM) was added into wells containing a fixed amount of DENV2 rNS2B3pro (100 μg/mL) and measured at 30 min. Figure shows relative fluorescence compared to Bz-nKRR-MCA under the same conditions. Error bars show standard deviation (SD). Figure S2B. Protease activity under different glycerol concentrations. Relative protease activity compared to 20 % glycerol standard. Error bars indicate SD. Figure S3A. Inhibition of protease activity by different aprotinin concentrations. Protease activity in the presence of control inhibitor; aprotinin was shown as percentage of enzyme only control. Concentrations ≤10 μM aprotinin significantly differ from the blank (buffer only) at 100 μg/mL recombinant protease concentration. Error bars indicate SD. Figure S3B. A fixed amount of DENV protease was subjected to various concentrations of an inhibitor. Aprotinin, showing trend over time. All sample reaction mixtures contain 100 μg/mL NS2B-NS3pro and 100 μM Bz-nKRR-MCA suspended in 200 mM Tris-HCl with 20 % glycerol, pH 9.5, unless otherwise specified. Error bars indicate standard deviation. (DOCX 1640 kb

Basic characteristics of participants.

<p>Basic characteristics of participants.</p

Region-specific, wavelike dynamics of the nidovirus genome expansions.

<p>Relative contributions of the genome regions ORF1a, ORF1b, 3′ORFs, 5′UTR and 3′UTR to the increase in genome size are calculated according to the splines regression and plotted on top of each other and against their sum = 1. Solid horizontal lines and vertical bars on top: genome size ranges and samplings for nidovirus lineages indicated by names. Dotted lines: topology of major nidovirus branches. Selected domains gained (ExoN, OMT, NMT, RsD and ADRP, circles) and lost (NendoU and NMT, diamonds) are colored according to ORF in which they are encoded. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003500#ppat-1003500-g003" target="_blank">Fig. 3</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003500#ppat.1003500.s002" target="_blank">Fig. S2</a> and the main text.</p

Comparison of regression models.

a<p>linear regression model (linear); double-logistic regression model (dlog); 3^rd order monotone splines regression model (splines).</p>b<p>standard weighted F test (F); permutation F test (F_perm); a weighted version of a test to compare non-nested regression models (LV) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003500#ppat.1003500-Lavergne1" target="_blank">[112]</a>.</p>c<p>shown is the probability that model A (null hypothesis) fits the data better than model B (alternative hypothesis); asterisks (*) highlight significant values to reject the null in favor of the alternative hypothesis using a confidence level of 0.05; probabilities are calculated separately for ORF1a, ORF1b, 3′ORFs as well as the complete model combining the three coding plus the two UTR regions (total).</p>d<p>none of the 1 million permutations resulted in an F larger than that of the non-permuted dataset.</p

Hierarchy and cooperation in the nidovirus genome expansions.

<p>Functional and evolutionary relations between the three major coding regions of the nidovirus genome are depicted. For a brief description on the relationship between these three coding regions and the processes they dominate in the nidovirus life cycle, see text.</p

Relationship of sizes of three major coding regions and genome size in the nidovirus evolution.

<p>For 28 nidoviruses representing species diversity, absolute sizes of 3′ORFs (A), ORF1a (B), and ORF1b (C) are plotted against the size of the genome. Different symbols were used to group the viruses into five major phylogenetic lineages (see inlet in A). Results of weighted linear, double-logistic and 3rd order monotone splines <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003500#ppat.1003500-Ramsay1" target="_blank">[111]</a> regression analyses are depicted. The three regression models (see inlet in C) fit the data with weighted r² values of 0.908 (linear), 0.949 (double-logistic) and 0.960 (splines) for ORF1a, 0.758, 0.898 and 0.929 for ORF1b, and 0.835, 0.950 and 0.954 for 3′ORFs. For fit comparison of regression models see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003500#ppat-1003500-t001" target="_blank">Table 1</a>.</p

Relationship of evolutionary distance to genome size change in nidoviruses.

<p>Evolutionary distance (average number of substitutions per amino acid position in the conserved proteins) in relation to difference in genome size is shown for each pair (n = 378) of the 28 nidovirus species. Points are colored according to pairs of major clades shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003500#ppat-1003500-g001" target="_blank">Fig. 1A</a>. The number of comparisons for each pair of clades is indicated by numbers in brackets. Points were grouped into clusters I (intra-lineage comparisons), II (large- vs. large-sized inter-lineage comparisons), III (intermediate-sized vs. others) and IV (small- vs. large-sized).</p

Nidovirus genome and region size differences.

<p>Shown are size distributions of genomes (left part) and the three genome coding parts ORF1b, ORF1a and 3′ORFs (right part) for five small-sized arterivirus species (small), 22 large-sized nidovirus species (large) and one intermediate-sized mesonivirus species (interm.). The distributions are represented by box-and-whisker graphs, where the box spans from the first to the third quartile and includes the median (bold line). The whiskers extend (dashed lines) to the extreme values.</p

Phylogeny of nidoviruses in comparison to the Tree of life (ToL).

<p>Bayesian phylogenies of nidoviruses (A) and ToL (B) are drawn to a common scale of 0.1 amino acid substitutions per position. Major lineages are indicated by vertical bars and names; arteri: <i>Arteriviridae</i>, mesoni: <i>Mesoniviridae</i>, roni: <i>Roniviridae</i>, toro: <i>Torovirinae</i>, corona: <i>Coronavirinae</i>. Rooting was according to either (A) domain-specific outgroups <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003500#ppat.1003500-Nga1" target="_blank">[10]</a> or (B) as described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003500#ppat.1003500-Boussau1" target="_blank">[66]</a>. Posterior probability support values and fixed basal branch points (*) are indicated. The nidovirus and ToL alignments include, respectively, three enzymes and 56 single-gene protein families, 604 and 3336 columns, 2.95% and 2.8% gaps. For further details on the nidovirus tree see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003500#ppat.1003500-Nga1" target="_blank">[10]</a>.</p

Viral loads and cytokine levels in the brains of severe and mild cases of JaOArS982-infected mice.

<p>(A) Viral loads in the CNS of severe (weight loss: <0.75, n=8) and mild (weight loss: >0.90, n=24) cases of JaOArS982-infected B6 mice at 13 days pi. P: Mann Whitney test. (B) mRNA levels of TNF-α, IL-10, IFNγ, IL-2, IL-4 and IL-5 quantified by real-time PCR in the brain cortex of JaOArS982-infected B6 mice at 13 days pi. Uninfected group (U group, n=8), Severe group: S (n=8), Mild group with high viral load of >10⁶ pfu/g of brain tissue: MH (n=11), Mild group with low viral load of <10⁶ pfu/g of brain tissue: ML (n=13). P: Kruskal-Wallis test, <i>p</i>: Mann Whitney test.</p