Tracking the Evolution of Dengue Virus Strains D2S10 and D2S20 by 454 Pyrosequencing

<div><p>Dengue virus is the most prevalent mosquito-borne virus worldwide. In this study, we used pyrosequencing to analyze the whole viral genome of two mouse-adapted strains, D2S10 and D2S20, that induce a dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)-like lethal disease in mice lacking the type I and/or type II interferon receptors. Previous experiments with D2S10 indicated that N124D and K128E mutations in the envelope protein were responsible for the severe disease induced in mice compared to its parental strain PL046. Here we demonstrate that D2S20 is more virulent than D2S10 and captured the presence of five key amino acid mutations – T70I, N83D, and K122I in envelope (E), and A62T in nonstructural protein 2A (NS2A) and G605V in nonstructural protein 5 (NS5) – that may account for this. These findings set the foundation for further dissection of the viral determinants responsible for dengue disease manifestations in mouse models.</p> </div

Common amino acid changes that occur in DENV2 strains D2S10 and D2S20 relative to the parental strain PL046, but are sustained with higher frequency in D2S20 than in D2S10.

<p>E, envelope; NS2A and NS5, nonstructural proteins 2A and 5. * Denotes a frequency above 50%.</p

Amino acid changes that are novel to DENV2 strain D2S20 relative to D2S10.

<p>M, membrane; NS3, NS4A, and NS4B, nonstructural protein 3, 4A, and 4B.</p

Mutation mapping of DENV2 strain D2S20.

<p>Red and green arrows indicate changes observed in D2S10 that are amplified in D2S20 in comparison to the DENV2 PL046; red arrows show the substitutions that occur at a frequency above 50%, while the green arrow indicates a frequency below 50%. Black arrows denote novel mutations found only in D2S20 that occur at a frequency above 1%. A blue arrow indicates a mutation that occurred at 100% frequency in both DENV2 strains D2S10 and D2S20 in comparison to the DENV2 PL046 isolate.</p

DENV replication in the liver and kidney after D2S10 and D2S20 infection.

<p>Viral RNA levels in (a) liver and (b) kidney were quantified by qRT-PCR 48 and 72 hours after infection (10¹⁰ GE of DENV2 strain i.v. on day 0) D2S10 (white circles) and D2S20 (black circles). P values from two-tailed unpaired t test with Welch's correction (95% confidence interval [CI]): * P is<b>_</b>0.05, and ** P is<b>_</b>0.01. Dashed lines denote limits of detection.</p

Survival of mice following infection with DENV.

<p>Survival was monitored for 30 days in 5–6 week old mice infected i.v. with DENV2 strain D2S10 or D2S20. (a) AG129 mice were inoculated with 10⁹ or 10¹⁰ GE (4–5 mice per group). Differences in survival between all groups were statistically significant (p<0.005) except between groups infected with D2S10 (p = 0.0139) using the log-rank test (4–5 mice per group). (b) IFNAR^−/− mice were inoculated with 10¹⁰ or 10¹¹ GE (6 mice per group). Statistically significant differences were observed between mice infected with 10¹¹ GE D2S10 and 10¹¹ GE D2S20 (p = 0.0040).</p

Effect of down-sampling Illumina coverage on true positive and false positive rates.

<p>Plots are of the median true positive and false positive minority variant detection rates of the control library with the standard deviation shown as error bars from 10 iterations of random sampling from all reads to generate each coverage depth.</p

Measured versus expected minority variant percentages detected in the control library by Illumina and 454 sequencing.

<p>(A) Nucleotide percentages are plotted with linear regression line and 95% confidence intervals. (B) Amino acid percentages are plotted with 95% confidence intervals. MV, minority variant.</p

Minority variants detected by Illumina and/or 454 sequencing in the 5 patient samples combined.

<p>(A) Nucleotide minority variants categorized by platform (Illumina vs. 454) and whether the minority variants were detected by both Illumina and 454 or by one platform only. The “Illumina (w/454)” category refers to the Illumina minority variant calls that are also detected by 454 and the “454 (w/Illumina)” category refers to the 454 calls that are also detected by Illumina. (B) Pearson correlation of the nucleotide minority variants detected by both Illumina and 454. (C) Amino acid minority variants categorized by platform and whether the variants were detected by both Illumina and 454 or by one platform only. (D) Pearson correlation of the amino acid minority variants detected by both Illumina and 454. MV, minority variant.</p

Performance of Illumina and 454 sequencing for the detection of amino acid minority variants within the control library.

<p>Expected variant percentages include positions where a MV is present on more than one clone. Median variant % reflects only the minority variants detected by each platform and does not include the undetected variants. N represents the number of nucleotide positions in the control library where the variant frequency is expected. Illumina detected 1 false positive minority variant present at 0.7% of the viral population and 454 detected 5 false positive minority variants ranging from 0.09% to 0.6%. FN, false negative.</p