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Antiviral activity of the EB peptide against zoonotic poxviruses.

Altmann SE, Brandt CR, Jahrling PB, Blaney JE - Virol. J. (2012)

Bottom Line: The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus.A scrambled peptide had no inhibitory activity against either virus.Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. altmannse@niaid.nih.gov

ABSTRACT

Background: The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for in vitro activity against the zoonotic orthopoxviruses cowpox and monkeypox and for in vivo activity in mice against vaccinia and cowpox.

Findings: In yield reduction assays, EB had an EC50 of 26.7 μM against cowpox and 4.4 μM against monkeypox. The EC50 for plaque reduction was 26.3 μM against cowpox and 48.6 μM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox in vitro by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no in vivo activity against cowpox in mice.

Conclusions: While EB did demonstrate some in vivo efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment in vitro than as a therapeutic against orthopoxviruses in vivo.

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Effect of EB on CPXV and MPXV attachment and entry. CPXV (A, B, C, D) or MPXV (E, F) in the presence (B, D, F) or absence (A, C, E) of 100 μM EB was allowed to attach to BSC-1 cells for 1 hr at room temperature. Samples for attachment staining (A, B, E, F) were fixed with 4% paraformaldehyde, quenched for 5 minutes with 100 mM glycine, blocked with 10% FBS in PBS, and stained with 1:200 anti-VACV antibody (V0500-11D, US Biologial). Samples for entry staining (C, D) were incubated 1 hr at 37°C prior to PFA fixation, glycine quenching, permeabilization with 0.1% saponin, and staining with anti-core antibody (R236). Nuclei were counterstained with DAPI (blue). Arrows indicate selected virions (green) or virus cores (red).
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Figure 3: Effect of EB on CPXV and MPXV attachment and entry. CPXV (A, B, C, D) or MPXV (E, F) in the presence (B, D, F) or absence (A, C, E) of 100 μM EB was allowed to attach to BSC-1 cells for 1 hr at room temperature. Samples for attachment staining (A, B, E, F) were fixed with 4% paraformaldehyde, quenched for 5 minutes with 100 mM glycine, blocked with 10% FBS in PBS, and stained with 1:200 anti-VACV antibody (V0500-11D, US Biologial). Samples for entry staining (C, D) were incubated 1 hr at 37°C prior to PFA fixation, glycine quenching, permeabilization with 0.1% saponin, and staining with anti-core antibody (R236). Nuclei were counterstained with DAPI (blue). Arrows indicate selected virions (green) or virus cores (red).

Mentions: Orthopoxvirus cores are only accessible to antibodies after being released into the cytoplasm, allowing for the differentiation between attached and entered virus using virion-specific or core-specific antibodies, respectively [11]. To examine whether EB was disrupting virus attachment or entry into the cells, immunofluorescent microscopy was used to quantify the number of attached and entered viruses per cell for 100 cells in the absence or presence of 100 μM EB. The presence of EB had no significant effect on the number of CPXV virions attached to cells (5.4 virions/cell, untreated vs.4.7 virions/cell, treated, p > 0.01; Figure 3Avs. 3B) but significantly reduced virus entry (1.3 cores/cell, untreated vs. 0.3 cores/cell, treated, p < 0.0001; Figure 3Cvs. 3D). These data are similar to what has been reported with VACV, where virus attachment was unaffected by peptide treatment but core release was significantly reduced [5]. In contrast, MXPV attachment was significantly inhibited by EB (3.2 virions/cell, untreated vs. 0.9 virions/cell, treated, p < 0.001; Figure 3Evs. 3F). Based on the observed disruption of virus attachment, MPXV entry was not examined. This different target of inhibition is consistent with the different pattern of EB susceptibility displayed by MPXV in the yield reduction assay compared to that demonstrated by CPXV and VACV. As inhibition of both CPXV and MPXV by EB occurs extracellularly, it is unlikely that the peptide's previously-described ability to inhibit NF-κB signaling [1] is involved in its anti-orthopoxvirus activity.


Antiviral activity of the EB peptide against zoonotic poxviruses.

Altmann SE, Brandt CR, Jahrling PB, Blaney JE - Virol. J. (2012)

Effect of EB on CPXV and MPXV attachment and entry. CPXV (A, B, C, D) or MPXV (E, F) in the presence (B, D, F) or absence (A, C, E) of 100 μM EB was allowed to attach to BSC-1 cells for 1 hr at room temperature. Samples for attachment staining (A, B, E, F) were fixed with 4% paraformaldehyde, quenched for 5 minutes with 100 mM glycine, blocked with 10% FBS in PBS, and stained with 1:200 anti-VACV antibody (V0500-11D, US Biologial). Samples for entry staining (C, D) were incubated 1 hr at 37°C prior to PFA fixation, glycine quenching, permeabilization with 0.1% saponin, and staining with anti-core antibody (R236). Nuclei were counterstained with DAPI (blue). Arrows indicate selected virions (green) or virus cores (red).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275487&req=5

Figure 3: Effect of EB on CPXV and MPXV attachment and entry. CPXV (A, B, C, D) or MPXV (E, F) in the presence (B, D, F) or absence (A, C, E) of 100 μM EB was allowed to attach to BSC-1 cells for 1 hr at room temperature. Samples for attachment staining (A, B, E, F) were fixed with 4% paraformaldehyde, quenched for 5 minutes with 100 mM glycine, blocked with 10% FBS in PBS, and stained with 1:200 anti-VACV antibody (V0500-11D, US Biologial). Samples for entry staining (C, D) were incubated 1 hr at 37°C prior to PFA fixation, glycine quenching, permeabilization with 0.1% saponin, and staining with anti-core antibody (R236). Nuclei were counterstained with DAPI (blue). Arrows indicate selected virions (green) or virus cores (red).
Mentions: Orthopoxvirus cores are only accessible to antibodies after being released into the cytoplasm, allowing for the differentiation between attached and entered virus using virion-specific or core-specific antibodies, respectively [11]. To examine whether EB was disrupting virus attachment or entry into the cells, immunofluorescent microscopy was used to quantify the number of attached and entered viruses per cell for 100 cells in the absence or presence of 100 μM EB. The presence of EB had no significant effect on the number of CPXV virions attached to cells (5.4 virions/cell, untreated vs.4.7 virions/cell, treated, p > 0.01; Figure 3Avs. 3B) but significantly reduced virus entry (1.3 cores/cell, untreated vs. 0.3 cores/cell, treated, p < 0.0001; Figure 3Cvs. 3D). These data are similar to what has been reported with VACV, where virus attachment was unaffected by peptide treatment but core release was significantly reduced [5]. In contrast, MXPV attachment was significantly inhibited by EB (3.2 virions/cell, untreated vs. 0.9 virions/cell, treated, p < 0.001; Figure 3Evs. 3F). Based on the observed disruption of virus attachment, MPXV entry was not examined. This different target of inhibition is consistent with the different pattern of EB susceptibility displayed by MPXV in the yield reduction assay compared to that demonstrated by CPXV and VACV. As inhibition of both CPXV and MPXV by EB occurs extracellularly, it is unlikely that the peptide's previously-described ability to inhibit NF-κB signaling [1] is involved in its anti-orthopoxvirus activity.

Bottom Line: The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus.A scrambled peptide had no inhibitory activity against either virus.Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. altmannse@niaid.nih.gov

ABSTRACT

Background: The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for in vitro activity against the zoonotic orthopoxviruses cowpox and monkeypox and for in vivo activity in mice against vaccinia and cowpox.

Findings: In yield reduction assays, EB had an EC50 of 26.7 μM against cowpox and 4.4 μM against monkeypox. The EC50 for plaque reduction was 26.3 μM against cowpox and 48.6 μM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox in vitro by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no in vivo activity against cowpox in mice.

Conclusions: While EB did demonstrate some in vivo efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment in vitro than as a therapeutic against orthopoxviruses in vivo.

Show MeSH
Related in: MedlinePlus