Mass spectrum of peptide 2 identified through MALDI-ToF mass spectrometry corresponding to m/z 1949.

<p>Pools of overlapping peptides (18-mers, n = 5) spanning the entire E protein of DENV-2 were assembled such that no pool contained peptides of similar mass. These peptide pools were allowed to react with IgG purified from DENV-2 infected patient’s sera and the antibody-bound peptides were extracted in acidic conditions. Following extraction, one peptide with a m/z value of 1949 was identified through MALDI-ToF mass spectrometry. MS/MS sequencing of peptide ion mass 1949 unequivocally defines it as deriving from peptide 2 sequence (NRDFVEGVSGGSWVDIVL) with the b and y ion coverage shown in the table.</p

Location of epitopes on the DENV-2 protein.

<p>Top view of the dimeric form of the DENV-2 E protein residues 1–395 (PDB ID-LOAN). The domains I, II and III are coloured red, yellow and blue, respectively. The epitopes identified from 3 different epitope mapping strategies were coloured in green. A) e pitope B2, B) epitope B16, C) epitope B19, D) epitope B29, E) epitope B38, F) epitope B45.</p

Comparison of amino acid sequences of peptides B2, B16, B29, B38, B45, B64 and B19 between the four DENV serotypes.

<p>Comparison of amino acid sequences of peptides B2, B16, B29, B38, B45, B64 and B19 between the four DENV serotypes.</p

Synthetic peptides of the DENV-2 E-glycoprotein binding with IgG in ELISA.

<p>The position of the 17 peptides (18-mers) recognized by the immune sera in ELISA is shown as grey bars. The soluble E protein (residues 1–395) comprising domains I, II, and III are coloured in red, yellow, and blue, respectively. The peptide sequences for each of the synthetic peptides are shown.</p

Neutralizing activity of anti-peptide antibodies against four DENV serotypes.

<p>Serially diluted pooled immune sera from vaccine constructs B16, B29, B38, B45, B64 and B19 were used to test the virus neutralizing activity against DENV-1, DENV-2, DENV-3 and DENV-4 in an <i>in vitro</i> focus reduction neutralization assay (FRNT) employing BHK-21 cells. A dilution resulting in 50% reduction of focus when compared to the pre-immune serum was considered as the end-point titre (FRNT₅₀). Each neutralization curve is an average (±S.E.M) of two independent neutralization experiments with a pooled serum group.</p

Immunogenicity of DENV E protein (PDB id: 1k4r) super imposed with Bayesian Predictive Algorithm.

<p>Top view of the homology model of DENV E protein monomer, PDB id- 1k4r is shown with amino acid residues indicated in space filling model. Colour coded regions correspond to epitopes predicted to have neutralizing ability. Areas shaded in red are indicative of most conserved, surface exposed residues and most likely immunogenic. Areas shaded in light pink are exposed and likely immunogenic, where as those in blue are considered least conserved and not immunogenic.</p

Epitopes identified on the DENV-2 E protein.

<p>Epitopes identified on the DENV-2 E protein.</p

Cross-reactive binding response of anti-peptide antibody against the sE recombinant protein of DENV-1, DENV-2 and DENV-3.

<p>Sera raised against each peptide group were pooled (n = 5) and the resulting immune sera were used to test the cross-reactive antibody response in ELISA. The log₁₀ antibody titres raised against each vaccine construct (B16, B29, B38, B45, B64 and B19) were evaluated using the recombinant E protein of DENV-1 (clear bar), DENV-2 (dark bar) and DENV-3 (shaded bar).</p

Inhibitory effects of plant extracts on H1N1 influenza virus.

<p>Cells at 80% confluency were treated with two-fold serial dilutions of plant extract (0.78–100 µg/mL) and 100 TCID₅₀ of H1N1 simultaneously. All wells were provided with 100 µL of RPMI medium supplemented with 2 µg/mL trypsin (virus growth medium). Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with <i>p</i><0.05 (one way ANOVA).</p

Inhibitory effects of anti-influenza extracts on the binding of H3N1 and H1N1 strains.

<p>The activity of extracts against the binding of influenza virus at a concentration of 50, 12.5 and 0.78 µg/mL is shown along with standard errors. Plant extracts were challenged with 200 TCID₅₀ of either of the two virus strains. A negative sign indicates lack of anti-influenza activity.</p