- Short report
- Open Access
Antiviral activity of the EB peptide against zoonotic poxviruses
© Altmann et al; BioMed Central Ltd. 2011
- Received: 2 November 2011
- Accepted: 6 January 2012
- Published: 6 January 2012
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.
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.
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.
- EB peptide
- poxvirus entry
- poxvirus attachment
The EB peptide (NH2- RRKKAAVALLPAVLLALLAP-COOH) is a 20-mer derived from the signal peptide of the human FGF4 protein  and was originally identified as an inhibitor of herpes simplex virus entry . Subsequent work demonstrated that EB was active against several strains of influenza virus both in vitro and in vivo , with a minimum of 13 core amino acids being identified as necessary to block influenza attachment to host cells . EB was also identified as an inhibitor of Vaccinia virus entry into host cells in vitro . This broad range of antiviral activity against a number of unrelated viruses, in combination with low in vivo toxicity , makes EB an attractive candidate for a broad-spectrum antiviral therapy.
Vaccinia virus (VACV) is the most-studied member of the orthopoxviruses, a genus of large, double-stranded DNA virus whose most notorious member, Variola virus, the etiologic agent of smallpox, was declared eradicated in 1980 . Vaccinia virus infection typically results in a self-limiting infection in immunocompetent individuals; the closely-related cowpox (CPXV) and monkeypox (MPXV) viruses, however, are both considered to be emerging zoonotic agents [8, 9] with the potential to cause serious morbidity and, in the case of MPXV, mortality in infected hosts . There are currently no FDA-approved therapeutics for treating orthopoxvirus infections, and vaccination is counter-indicated for an increasingly large percentage of the global population, highlighting the need for novel therapeutic options. The relatively low global incidence of severe orthopoxvirus disease, however, makes identifying broad spectrum drugs with activity against a number of unrelated viruses, including the orthopoxviruses, economically advantageous. To expand upon the initial characterization of EB peptide anti-orthopoxvirus activity, the goals of this work were to test EB for efficacy against CPXV and MPXV in vitro, to begin to determine the mechanism for any inhibition observed, and to test EB for in vivo activity in two well-characterized mouse models of orthopoxvirus disease, VACV and CPXV.
In summary, we were able to demonstrate in vivo efficacy of EB against VACV but not CPXV, despite both viruses having similar sensitivities to the peptide in vitro. The reasons for this difference are as yet unclear. EB was only effective against VACV when co-administered with the virus, suggesting that the peptide needed direct interaction with the virus to be effective. EB self-associates in micelle-like structures at high concentrations and in high ionic buffers , a property which could influence bioavailability. It is possible that the in vivo anti-orthopoxvirus activity could therefore be improved by changing the vehicle used for delivery.
Most intriguing is the observation that EB inhibited attachment by MPXV but blocked entry by CPXV, as these processes are generally believed to be highly conserved between the orthopoxviruses. While EB has now been shown to have activity in vivo against VACV and influenza viruses , its greatest potential with orthopoxviruses may be as a novel in vitro tool to study the poorly-characterized early steps in infection. To date, over a dozen viral proteins are believed to be involved in orthopoxvirus attachment and entry [13–25]. The precise mechanisms of attachment and entry, however, have yet to be elucidated. Identification of the precise targets of the interaction between EB and VACV, CPXV, and MPXV could help identify key amino acids or structural features necessary for these processes and identify targets for novel inhibitors of infection.
The authors thank Grant McFadden (University of Florida) for the gift of the cowpox-GFP virus and Roselyn Eisenberg and Gary Cohen for the R236 anti-core antibody. The authors thank Russ Byrum, Isis Alexander and Bernardo Rosa for technical assistance. This work was supported in part by the NIAID Division of Intramural Research and by grants AI52049 and EYP30-016665 to Curtis R. Brandt. All animal work was performed under a NIAID-approved protocol and in compliance with NIAID's Institutional Animal Care and Use Committee.
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