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Results: 7

1.
Figure 7

Figure 7. Proposed locations of the SP40 peptide based on sequence alignment and molecular modeling of poliovirus structure.. From: Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1.

(A) The EV-71 strain 41 was aligned with Mahoney poliovirus strain using Clustal W2 program and (B) The molecular structure of poliovirus VP1, VP2, VP3, and VP4 is represented by purple, blue, brown and green, respectively. The SP40 sequence is indicated in yellow.

Chee Wah Tan, et al. PLoS One. 2012;7(5):e34589.
2.
Figure 6

Figure 6. Alanine scanning analysis.. From: Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1.

Alanine scanning was performed on the SP40 peptide. Thirteen different peptides were synthesized by replacing one residue at a time with an A and their inhibitory effect was determined as described above. Activity of each peptide was compared with the wild-type SP40, which was represented by the red line. Numbers higher than the red line showed a gain of activity whereas a lower number represented a loss of activity.

Chee Wah Tan, et al. PLoS One. 2012;7(5):e34589.
3.
Figure 4

Figure 4. The antiviral activities of the SP40 peptide in various cell lines.. From: Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1.

Vero, HeLa and HT-29 cell lines were pre-treated with the SP40 peptide at various concentrations for 1 hour at room temperature before infection with EV-71 at a MOI of 0.1. (A) The viral induced cytopathic effects in various cell lines were observed 24-hour post-infection. (B) The viral RNA inhibition that were quantitated by RT TaqMan real-time PCR assay.

Chee Wah Tan, et al. PLoS One. 2012;7(5):e34589.
4.
Figure 1

Figure 1. Identification of antiviral peptides.. From: Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1.

A library consisting of 95-overlapping peptides (15 mers) covering the entire EV-71 capsid protein, VP1 was synthesized. Each peptide was screened at a presumptive concentration of 100 µM for its ability to inhibit EV-71 infection in plaque reduction assay. The top line showed a schematic representation of the EV71 genome, and the activities of the peptides were shown at the bottom. Arrows denote the four peptides that were positive in the assay.

Chee Wah Tan, et al. PLoS One. 2012;7(5):e34589.
5.
Figure 3

Figure 3. Antiviral activities of the SP40 and SP40X peptides.. From: Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1.

Both RD cells and EV-71 were separately pre-incubated with increasing concentrations of each peptide for 1 hour before viral inoculation. The inhibitory levels of the peptides were evaluated at 24-hour post-infection by (A) plaque assay and (B) RT TaqMan real-time PCR. (C) Antiviral properties of the SP40 peptide against different EV-71 strains in the comprehensive assay and (D) Mechanism of action studies: The SP40 peptide was added to EV-71 infection at different time points relative to viral inoculation as previously described in the Materials and Methods.

Chee Wah Tan, et al. PLoS One. 2012;7(5):e34589.
6.
Figure 2

Figure 2. Inhibitory effects of SP40 and SP40X peptides in cytopathic effect, plaque formation and protein synthesis.. From: Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1.

(A) For cytopathic effect, EV-71 at a MOI of 0.1 was pre-incubated with peptides for 1 hour before infection of peptide-treated RD cells. The images were taken at 24-hour post-infection. (B) For plaque reduction assay, approximately 100 PFU of EV-71 were pre-incubated with peptides for 1 hour before infection of peptide-treated RD cells. The cells were fixed with 4% formaldehyde and stained with 0.5% crystal violet at 48-hour post-infection. (C) Western blot analysis of total protein isolated from virus-infected cells using the EV-71 monoclonal antibody (Millipore, Billerica, USA) and monoclonal anti β-actin antibody (Sigma, St. Louis, USA).

Chee Wah Tan, et al. PLoS One. 2012;7(5):e34589.
7.
Figure 5

Figure 5. Viral attachment assay.. From: Inhibition of Enterovirus 71 (EV-71) Infections by a Novel Antiviral Peptide Derived from EV-71 Capsid Protein VP1.

(A) RD cells were grown in the chamber slides (Lab-tek, Rochester, USA) and incubated at room temperature for 1 hour with or without the SP40 peptide. This was followed by the incubation of the cells in the cold with EV-71 for 1 hour and washing off the unbound virions with PBS. RD cell monolayers were fixed with 4% paraformaldehyde and subsequently blocked with Image-iT™ FX Signal Enhancer (Invitrogen, San Diego, USA). The EV-71 particles were probed with anti-EV-71 monoclonal antibody (Millipore, Billerica, USA) and Alexa Fluor 488 anti-mouse IgG (Invitrogen, San Diego, USA). The nuclei were stained with DAPI for 7°minutes at room temperature. The images were obtained from the fluorescent microscopy. EV-71 viral particles and cell nuclei were shown in green and blue fluorescence, respectively. The number of virus particles that was attached to the cell surface were quantitated by (B) Cellomics HCS ArrayScan Spot Detector Bio-Application and (C) RT TaqMan real-time PCR assay

Chee Wah Tan, et al. PLoS One. 2012;7(5):e34589.

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