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I. Galindo et al. / Antiviral Research 91 (2011) 57–63
In order to examine the precise stage of the virus cycle blocked
Acknowledgments
by stilbenes, we analyzed the presence of specific early and late
proteins induced by ASFV in the presence or absence of the drugs.
The synthesis of late protein p72 was markedly decreased while
that of the early protein p30 was not affected. These findings
strongly suggest that the early phases of ASFV replication are not
targets for the antiviral effect of resveratrol or oxyresveratrol. As
early infection steps are not affected, a later infection step, neces-
sary for viral replication and subsequent infection rounds, should
be impaired by these antivirals. Moreover, both resveratrol and
oxyresveratrol had similar pattern of inhibition.
Quantitative PCR analysis showed an approximately 50%
decrease in viral genome copy numbers in Vero cells infected in
presence of 1 lg/ml of resveratrol or 5 lg/ml of oxyresveratrol.
Thus, resveratrol and oxyresveratrol inhibited viral DNA replica-
tion, subsequently inhibiting late viral protein synthesis.
Protein p54 is a late virus protein essential for virus replication
and it is incorporated into the external envelope of virions
(Rodriguez et al., 1994, 1996). B54GFP-2 is an infectious recombi-
nant ASFV that expresses and incorporates a chimera of the p54
protein fused to the enhanced green fluorescent protein (EGFP)
into the virus particle. Like p54, B54GFP-2 shows an intracellular
distribution that is fully coincident with the ASFV replication site,
also called the viral factory, at late post-infection times (Hernáez
et al., 2006). The ASFV replication site is characteristically found
in a perinuclear area, coincident with the MTOC. We studied the
formation of viral factories in Vero cells infected with Ba54GFP-2
in the absence and presence of resveratrol and oxyresveratrol. Both
drugs significantly reduced the number of viral factories and mod-
ified morphology and localization of viral replication sites, which
appeared dispersed. This finding indicates that the organization
and formation of viral replication sites results was impaired by
these antivirals.
There is an urgent need for countermeasures against ASFV.
While vaccination is the first option in the prevention of animal
viral diseases, other alternatives, such as antiviral agents, are start-
ing to gain relevance in the control and eradication of these dis-
eases. Despite the intense effort to find a vaccine for ASFV, none
is yet available and the control of the disease relies on the early
diagnosis and ‘‘stamping-out’’ of the entire pig population in farms
in affected areas. In this scenario, there is an urgent need for alter-
natives and research should be oriented to antiviral research.
When an outbreak of ASF occurs, one option could be to apply
antiviral prophylaxis to treat pigs in farms located close to the in-
fected farm in order to create a ‘‘safety ring’’ around the outbreak
focus and thus control the spread of the infection. Stabilizing the
epidemic area by means of an antiviral agent may give the author-
ities time to organize the appropriate countermeasures and to re-
duce the number of animals to be slaughtered.
This work was supported by grants from the Spanish Ministry of
Science and Innovation Program Consolider CSD2006-00007, CSD
CTQ2009-12216/BQU, AGL2009-09209, Fundación Séneca-CARM
Project 08661/PI/08, UE Project EPIZONE FOOD-CT2006-016236
Welcome Trust Foundation WT075813 and FEDER Funds
OP2007-13. J. Berná and J. Fenoll thank the MICINN for a Ramón
y Cajal contract.
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Our results demonstrate a potent, dose-dependent anti-ASFV
effect of resveratrol and oxyresveratrol. We propose that mulberry
twigs, which contain large amounts of oxyresveratrol, provide a
new natural source of starting material for the development of
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ASFV infection.