Research in Veterinary Science 88 (2010) 339–344

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Research in Veterinary Science

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Antiviral efficacy of EICAR against canine distemper virus (CDV) in vitro

Dal Pozzo Fabiana 1, Galligioni Viola, Vaccari Francesca, Gallina Laura, Battilani Mara, Scagliarini Alessandra *
Department of Veterinary Public Health and Animal Pathology, Alma Mater Studiorum, Università di Bologna via Tolara di Sopra 50, 40064 Ozzano Emila, Bologna, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Canine distemper virus (CDV) is a highly contagious pathogen of carnivores. In dogs, the disease is char-
Accepted 20 August 2009 acterized by high lethality rates and no specific antiviral therapy is available. The aim of this study was to
verify the in vitro antiviral activity of the 5-ethynyl-1-b-D-ribofuranosylimidazole-4-carboxamide (EICAR)
and to compare it with the 1-(b-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin, RBV).
Keywords: EICAR was more active than RBV against CDV replication, while both molecules exhibited low selectiv-
Canine distemper virus ity indexes. A reversal of their antiviral activity was observed after addition of guanosine, suggesting their
Paramyxoviridae
involvement in the inhibition of the inosine monophosphate dehydrogenase enzyme (IMPDH). RBV and
Antiviral activity
Ribavirin
EICAR had a time- and concentration-dependent anti-CDV activity, mainly displayed during the first 10 h
EICAR post-infection. The involvement of the inhibition of the viral RNA-dependent RNA polymerase (vRdRp) is
Mechanism of action discussed, as well as the role of CDV as a model to study more potent and selective antiviral molecules
active against other Paramyxoviridae.
Ó 2009 Published by Elsevier Ltd.

1. Introduction
Canine distemper (CD) is a highly contagious disease of domes-
tic and wild canids as well as of other terrestrial and aquatic carni-
vores (Appel, 1987; Osterhaus et al., 1988). CD is characterized by a
systemic infection with high lethality rates in hosts without pro-
tective immunity. The targets of infection are mainly mucous
membranes and lymphoid tissues throughout the body. The com-
mon manifestations of CD are pyrexia, anorexia, nasal discharge,
conjunctivitis, diarrhoea and skin pustules with hyperkeratosis
(Appel, 1987). Neurological symptoms appear during, after or in
the absence of the systemic phase of the disease and are mostly
responsible for the death of affected dogs (Vandevelde and
Zurbriggen, 1995). Canine distemper virus (CDV) is also the causa-
tive agent of old dog encephalitis (ODE), a rare chronic encephalo-
myelitis of mature dogs which has been compared to the subacute
sclerosing panencephalitis (SSPE) caused in humans by measles
virus (MV) (Vandevelde et al., 1980). Compelling similarities have
supported the use of the dog and ODE as a model in the study of
SSPE in humans (Vandevelde et al., 1980).
* Corresponding author. Address: Dipartimento di Sanità Pubblica Veterinaria e
Patologia Animale, Alma Mater Studiorum, Università di Bologna, via Tolara di
Sopra 50, 40064 Ozzano Emilia, Bologna, Italy. Tel.: +39 512097083; fax: +39
512097039.
E-mail address: alessand.scagliarini@unibo.it (A. Scagliarini).
1 protocols for the treatment of SSPE in humans (Hosoya et al.,
Current address: Department of Infectious and Parasitic Diseases, Virology and
Viral Diseases, University of Liège, Boulevard de Colonster, 20, B43b, b-4000, Liège,
Belgium.
Although the disease has generally been controlled with live
attenuated vaccines, outbreaks of CD are still reported worldwide
(Martella et al., 2008). To date, no effective antiviral treatment
exists despite an increasing demand from practitioners and dog
owners. In addition to therapy in pet animals, other possible uses
of anti-CDV drugs could be the treatment of dogs in kennels, of
expensive and valuable animals used in breeding programs and
of wild endangered animals in parks and zoologic gardens.
A prerequisite for the identification and rational design of
antiviral agents is knowledge of the viral biology. Antivirals can be
designed to interfere with viral entry or with the targets of the
post-entry viral life cycle. The replicative complex of paramyxovi-
ruses represents an attractive target in the development of
antiviral molecules, because human and animal tissues lack a known
homologue of the RNA-dependent RNA polymerase (RdRp) (Bourhis
et al., 2006). Despite the critical role in viral replication, the mecha-
nistic understanding of the paramyxovirus RdRp complex is still
rather poor. Several small compounds have been synthesised to tar-
get the RdRp complex, but no clinical trials have been conducted
(Sun et al., 2007; White et al., 2007; Yoon et al., 2008).
The nucleoside analogue 1-(b-D-ribofuranosyl)-1,2,4-triazole-3-
carboxamide (ribavirin, RBV) is the only commercially available
molecule with a well-known antiviral activity towards several
members of the Paramyxoviridae family (De Clercq et al., 1991;
Shigeta et al., 1992; Leyssen et al., 2005). Despite its low selectivity
and divergent results, RBV has been used in several experimental
2004; del Toro-Riera et al., 2006). RBV has pleiotropic modes of
action, including: (1) inhibition of the cellular enzyme inosine

0034-5288/$ - see front matter Ó 2009 Published by Elsevier Ltd.

doi:10.1016/j.rvsc.2009.08.010
340 F. Dal Pozzo et al. / Research in Veterinary Science 88 (2010) 339–344
monophosphate dehydrogenase (IMPDH) resulting in the deple-
tion of the intracellular guanosine-50 -triphosphate (GTP) pool
available for viral replication (Leyssen et al., 2005); (2) interference
with viral transcription by the inhibition of the capping enzymes
(Benarroch et al., 2004); (3) direct inhibition of the viral RNA poly-
merase (Maag et al., 2001); (4) incorporation into the viral genome
with subsequent lethal mutations (Crotty et al., 2001); and (5)
immunomodulation with increased interferon release by the in-
fected cells (Fang et al., 2000). Although the activity of RBV against
CDV has previously been reported (Scagliarini et al., 2006; Elia
et al., 2007), its mechanism of action has not yet been investigated.
In this study, our aim was to test the anti-CDV activity of the 5-
ethynyl-1-b-D-ribofuranosylimidazole-4-carboxamide (EICAR), an
analogue of RBV having a more potent antiviral activity against
several paramyxoviruses (De Clercq et al., 1991; Shigeta et al.,
1992). In an attempt to investigate their mechanisms of action, sev-
eral experiments have been conducted, mostly focussing on their
activity on the IMPDH and on the viral RdRp. Our results were com-
pared to the known activity of these molecules against other para-
myxoviruses in order to consider the possible use of CDV as a model
in studying the efficacy of more selective antiviral molecules.
2. Methods
2.1. Cells and virus
Ò
VERO cells (culture collection ATCC number CCL-81 ) were TM
used for the in vitro growth of canine distemper virus (CDV). VERO
cells were cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco’s
modified Eagle’s medium (Gibco, Invitrogen Corporation) supple-
mented with 10% fetal calf serum (FCS), 2 mmol/L of L-glutamine,
1 mmol/L of sodium pyruvate and 7.5% sodium bicarbonate.
Two cell-adapted strains of CDV were used: Bussell-CDV and
Ondestepoort-CDV (Haig, 1948; Bussell and Karzon, 1965). Both
CDV strains were propagated in VERO cells using a 2% FCS medium
and were subsequently titrated. The viral titre was expressed as
the 50% infectious dose of the tissue culture (TCID50/mL).
2.2. Antiviral molecules
The 5-ethynyl-1-b-D-ribofuranosylimidazole-4-carboxamide (EI-
CAR) (kindly provided by Prof. Robert Snoeck, Rega Institute for
Medical Research, KU Leuven, Belgium) and the 1-(b-D-ribofurano-
syl)-1,2,4-triazole-3-carboxamide (ribavirin, RBV) (ICN Biomedi-
cals) were tested against CDV.
2.3. Cytotoxicity assay
Both RBV and EICAR cytotoxicities were compared using a col-
orimetric assay based on the mitochondria metabolisation of the
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide
(MTT) (Sigma) (Denizot and Lang, 1986). The toxicity of the com-
pounds was measured on VERO cells in replication, as previously
described (Scagliarini et al., 2006). The 50% cytotoxic concentration
(CC50) was defined as the compound concentration required to re-
duce the intensity of the colour emission by 50% of the cell control.
The MTT assay was also used to evaluate RBV and EICAR toxicity on
stationary VERO cells. At least six independent experiments were
performed and the mean ± the standard deviation (SD) of the
CC50 values was calculated for RBV and EICAR.
2.4. Antiviral activity assay
EICAR and RBV antiviral activity towards Bussell-CDV and
Ondestepoort-CDV was evaluated with a cytopathic effect reduc-
tion assay (CPE-reduction assay) on confluent VERO cells, using a
protocol previously described (Scagliarini et al., 2006). The 50%
inhibitory concentration (IC50) was defined as the compound con-
centration required to reduce viral CPE by 50% of the virus control.
The IC50 values of RBV and EICAR were calculated as the mean ± SD
from at least six independent experiments. The selectivity index
(SI) was obtained by calculating the ratio of the cytotoxicity and
the antiviral activity values. The SI was calculated in stationary
and in growing VERO cells.
2.5. Antiviral activity in the presence of nucleosides
To evaluate the inhibition of the IMPDH enzyme during CDV
replication in VERO cells, a standard assay was performed with
RBV and EICAR in the presence of different concentrations of
nucleosides (guanosine, adenosine, cytidine and uridine) in the
culture medium. Each nucleoside had previously been tested alone
to exclude an inhibitory effect towards viral growth and was sub-
sequently tested in two different concentrations (10 and 100 lg/
mL) in the presence of RBV and EICAR. IC50 values were calculated
and expressed as the mean ± SD from at least three independent
experiments.
2.6. Virus yield assay
The effect of several dilutions of RBV and EICAR was evaluated
on the virus yield of CDV in VERO cells. VERO cells were grown in
6-well microtitre plates and the confluent monolayer was infected
with Bussell-CDV at a multiplicity of infection (MOI) of approxi-
mately 0.05. After 2 h incubation at 37 °C and in a 5% CO2 atmo-
sphere, any residual virus was removed and replaced by medium
containing different concentrations of the compounds tested (80,
40, 20, 10 lg/mL of RBV and 20, 10, 5, 2 lg/mL of EICAR). Infected
non-treated and mock infected controls were included at each time
point. At 24, 48 and 72 h post-infection (PI), the supernatants were
harvested and frozen at 80 °C; cell monolayers were also frozen
at 80 °C after adding PBS into each well. Collecting the superna-
tants and the cell monolayers separately allowed the quantifica-
tion of the extracellular and the intracellular viruses. In
particular, both samples were first used in a titration assay and
secondarily in a real time reverse transcription polymerase chain
reaction (RT-PCR). Titration was performed with serial 10-fold
dilutions of the samples and the viral titre was expressed in
TCID50/mL. A one-step real time RT-PCR was applied to the quanti-
fication of the viral genome. RNA samples were purified using
NucleoSpinÒ RNA II (Macherey–Nagel GmbH & Co. KG). TaqMan-
based RT-PCR was performed as previously described (Scagliarini
et al., 2007). Immediately before setting up each PCR reaction,
10-fold dilutions of an internal control were prepared and a stan-
dard curve was constructed. Each sample and each dilution of
the internal control were repeated in duplicate during the same
reaction and the RNA concentration (copies/lL) was calculated as
the mean of the two measurements.
2.7. Time of addition assay
Confluent VERO cells seeded into 96-well microtitre plates were
infected with Bussell-CDV at an MOI of 0.1. After an incubation
time of 2 h at 37 °C in a 5% CO2 atmosphere, the viral inoculum
was removed and replaced by 100 lL of 2% FCS culture medium.
At different time PI (2, 4, 6, 8, 10, 12, 16, 20, 24 and 48 h), 100 lL
of RBV and EICAR dilutions were added in duplicate to the micro-
plates. At each time point, virus and cell controls were constantly
included in the assay. In the presence of 100% CPE in the virus con-
trols, CPE formation was examined in relation to the dilution of the
compounds at the different time-points. The inhibition of CDV
 F. Dal Pozzo et al. / Research in Veterinary Science 88 (2010) 339–344
growth by the two compounds was expressed as the IC50 value at
each time point.
3. Results
3.1. Cytotoxicity and antiviral activity
The results obtained from the cytotoxicity and the antiviral
activity assays are shown in Table 1. EICAR had anti-proliferative
effects towards VERO cells in the growing phase whereas it was
not cytotoxic for stationary cells. EICAR was 9-fold more toxic than
RBV with a CC50 value of 3.6 ± 1.1 lg/mL as compared to 27.2 ±
2.4 lg/mL for RBV.
The antiviral activity of the two compounds did not differ
among the two CDV strains. Thus, in Table 1, the results of the anti-
viral activity are shown for Bussell and Ondestepoort, but also as
the average of the IC50 values of the two strains. RBV showed lower
antiviral activity with an average IC50 value of 25.5 ± 2.1 lg/mL as
compared to an average IC50 value of 4.6 ± 0.3 lg/mL for EICAR.
Both compounds were seen to be selective only in stationary cells
(RBV SI > 3.9, EICAR SI > 10).
3.2. Antiviral activity in the presence of nucleosides
None of the nucleosides revealed antiviral activity against CDV
(data not shown). The effects of several nucleosides on the antiviral
activity of RBV and EICAR are shown in Table 2. The addition of 10
and 100 lg/mL of adenosine, cytidine, and uridine to the culture
medium did not alter the antiviral activity of RBV and EICAR. When
10 lg/mL of guanosine were added into the culture medium, the
IC50 values of RBV and EICAR were completely cancelled. To clarify
the reversal of the antiviral effects of RBV and EICAR following the
addition of guanosine, additional concentrations of this nucleoside
were tested (0.5, 2, 5, 10 and 20 lg/mL). As shown in Table 2, the
presence of 5, 10 and 20 lg/mL of guanosine completely subverted
the IC50 values of RBV and EICAR. Lower concentrations of guano-
sine partially reversed the antiviral activity of the two molecules.
3.3. Virus yield assay
The reduction of viral growth in the presence of several concen-
trations of RBV and EICAR and at different times PI was evaluated
after viral titration and RNA quantification. Both techniques were
used to measure the virus yield from the supernatants and the cell
monolayers collected separately during the experiment. At each
time point, EICAR showed a higher anti-CDV activity than RBV. In
the supernatants after 24 h PI, viral growth was observed only in
the infected non-treated samples. At this time point, the lowest
concentrations of RBV and EICAR could completely inhibit the
growth of the extracellular virus (Fig. 1A and B). In the cell mono-
layers after 24 h PI, RBV and EICAR completely inhibited viral
growth up to 40 and 5 lg/mL, respectively. After 48 h PI, with
Table 1
Cytotoxicity and antiviral activity of RBV and EICAR.
Compounds Cytotoxicity Antiviral activity
a b
CC50 (lg/mL) IC50 (lg/mL)
Stationary cells Growing cells Bussell-CDV
RBV >100 27.2 ± 2.4 27 ± 7.5
EICAR >50 3.6 ± 1.1 4.8 ± 1.5
a
The values are expressed as the mean ± SD of the CC50 values after at least six independent experiments.
b
The values are expressed as the mean ± SD of the IC50 values after at least six independent experiments.
c
The mean of the IC50 values of the Bussell- and Ondestepoort-CDV strains.
d
SI = CC50/IC50.
341
80 lg/mL of RBV a total inhibition of viral growth was observed
in the supernatant. Likewise with 20 and 10 lg/mL of EICAR, no
virus was detected in the supernatants and a reduction of 2 and
1 Log10 TCID50/mL as compared to the control virus was observed
in the cell monolayers. After 72 h PI, in both the supernatants
and the cell monolayers, a decrease of 3 and 2 Log10 TCID50/mL
was calculated in the presence of 80 lg/mL of RBV and 20 lg/mL
of EICAR, respectively (Fig. 1C and D).
The reduction of the total viral RNA was expressed as RNA cop-
ies/lL (Fig. 2). In the supernatants, CDV nucleic acid could be de-
tected only starting at 48 h PI, at which time a complete
inhibition of the viral RNA synthesis emerged in the presence of
the highest concentrations of RBV and EICAR. At the last time point,
a reduction of 2–3 Log10 RNA copies/lL was calculated with 80 and
20 lg/mL of RBV and EICAR, respectively (Fig. 2A and B). The re-
sults of the virus yield assay showed that the reduction of viral
growth and the inhibition of the RNA synthesis induced by the
two molecules followed the same time- and concentration-depen-
dent trend.
3.4. Time of addition assay
The results obtained from the time of addition assay are shown
in Fig. 3. Between 2 and 8 h PI, the IC50 values measured for RBV
and EICAR were of the same magnitude as the antiviral assay
(ranging from 23.4 to 34.4 lg/mL for RBV and 2.4 to 5 lg/mL for EI-
CAR). For both molecules starting from 10 h PI, an increase of the
IC50 values was measured. After 20 h PI, the addition of RBV and EI-
CAR to the infected culture did not modify viral growth as com-
pared to the control virus (IC50 values > 100 lg/mL for RBV
and > 20 lg/mL for EICAR).
4. Discussion
In this study, we present original data on the antiviral activity of
EICAR against CDV and on its possible mechanisms of action. We
also compared the antiviral activity and the mechanism of action
of the compound with RBV. This work integrates the preliminary
data published by Scagliarini et al. (2006) on RBV anti-CDV activity,
adding the use of EICAR known for its higher antiviral activity
against several paramyxoviruses. In vitro experiments have been
conducted in the attempt to elucidate the multiple mechanisms
of action known for RBV and its analogue EICAR. Furthermore,
the results point out the possible use of CDV as a model in the
study of active molecules against members of the Paramyxoviridae
family.
First, we observed that EICAR had an anti-proliferative activity
towards VERO cells in replication as observed for RBV, confirming
previous observations in different cell lines and primary cultures
(De Clercq et al., 1991; Shigeta et al., 1992). EICAR showed a 5-fold
higher antiviral activity than RBV against CDV, but also an in-
creased cytotoxicity as previously shown against MV and other
SId
c
Average Average
Ondestepoort-CDV Stationary cells Growing cells
24 ± 7.2 25.5 ± 2.1 >3.9 1.07
4.4 ± 1.4 4.6 ± 0.3 >10 0.78
342 F. Dal Pozzo et al. / Research in Veterinary Science 88 (2010) 339–344

Table 2
Antiviral activity of RBV and EICAR following the addition of different nucleosides.

Nucleosides (lg/mL)a RBV antiviral activity IC50 (lg/mL)b EICAR antiviral activity IC50 (lg/mL)b
Bussell Ondestepoort Bussell Ondestepoort
No nucleosides 27 ± 7.5 24 ± 7.2 4.8 ± 1.5 4.4 ± 1.4
Adenosine 10 36.9 ± 3.7 ND 4.5 ± 0.5 ND
Adenosine 100 29.1 ND 4 ± 0.3 ND
Cytidine 10 33.3 ± 3 ND 4.2 ± 1.1 ND
Cytidine 100 31.2 ± 8.8 ND 4.8 ± 1 ND
Uridine 10 33.3 ± 3 ND 3.9 ± 0.5 ND
Uridine 100 34.3 ± 4.4 ND 4.3 ± 0.8 ND
Guanosine 0.5 47.2 ± 16.8 ND 12 ± 3.2 ND
Guanosine 2 86.1 ± 12.7 ND 32 ± 7.5 ND
Guanosine 5 >100 >100 >50 >50
Guanosine 10 >100 >100 >50 >50
Guanosine 20 >100 >100 >50 >50

ND: not determined.

a
The nucleosides were added at 10 and 100 lg/mL except for the guanosine experiments in which more concentrations were used (0.5, 2, 5, 10 and 20 lg/mL).
b
The values are expressed as the mean ± SD of the IC50 values after at least three independent experiments.

A Ribavirin B EICAR
100000
100000

10000 10000
TCID50/mL

TCID50/mL

1000 1000

100 100

10 10

1 1
0 10 20 40 80 CC 0 2 5 10 20 CC
concentration µg/mL concentration µg/mL

24 H 48 H 72 H 24 H 48 H 72 H

C 1000000 Ribavirin D EICAR

100000
100000
10000
10000
TDCI50/mL

TDCI50/mL

1000
1000
100
100

10 10

1 1
0 10 20 40 80 CC 0 2 5 10 20 CC
concentration µg/mL concentration µg/mL

24 H 48 H 72 H 24 H 48 H 72 H

Fig. 1. CDV titration following the virus yield assay. Viral growth was measured in the presence of different concentrations of RBV (A and C) and EICAR (B and D) after 24 h
(black columns), 48 h (grey columns) and 72 h PI (white columns). CDV infectivity was detected in the supernatants (A and B) and in the cell monolayers (C and D). At each
time point, non-infected (cell control, CC) and infected non-treated controls (0 lg/mL) were included. The viral titre was expressed in TCID50/mL and subsequently
represented on a Log scale.

paramyxoviruses (De Clercq et al., 1991; Shigeta et al., 1992; Leys-
sen et al., 2005). Neither molecule had any selectivity for VERO
cells in replication while a modest selectivity against CDV was ob-
served on stationary cells. Confluent cells were used in the follow-
ing experiments as the best condition to define the antiviral
activity of RBV and EICAR towards CDV.
Recently, it has been reported that RBV was active against CDV
in vitro (Scagliarini et al., 2006; Elia et al., 2007). Elia et al. (2007)
hypothesized that error catastrophe could be a possible mecha-
nism of action of RBV against CDV replication. This mechanism is
usually observed with high concentrations of RBV (Crotty et al.,
2001) which are difficult to use in the therapy due to the known
in vitro and in vivo cytotoxicity of the molecule. For this reason,
we have investigated the involvement of other mechanisms of ac-
tion, compatible with the use of lower concentrations of RBV. In
our study, we showed a reversal of RBV and EICAR antiviral activity
after the addition of guanosine, leading to the hypothesis that the
two molecules could act via inhibition of the cellular enzyme
IMPDH. This cellular target could justify the high cytotoxicity
and low selectivity of the two compounds. The role of IMPDH inhi-
 F. Dal Pozzo et al. / Research in Veterinary Science 88 (2010) 339–344
A Ribavirin
100000
10000
RNA copies/µL
1000
100
10
1 1
0 10 20
concentration µg/mL
48 H 72 H
Fig. 2. CDV nucleic acid quantification following the virus yield assay. Real time PCR was carried out in the presence of different concentrations of RBV (A) and EICAR (B). Total
viral RNA present in the supernatants collected after 48 h (grey columns) and 72 h PI (white columns) was measured. At each time point, non-infected (cell control, CC) and
infected non-treated controls (0 lg/mL) were included. The total RNA was expressed in copies/lL and represented on a Log scale.
120
Ribavirin EICAR
100
Ribavirin IC50 values
80
(µg/mL)
60
40
20
0 0
0 2 4 6 8 10 12 16 20
Time of addition (H)
Fig. 3. Time of addition assay. The illustration shows the IC50 values calculated after
the addition of RBV and EICAR into an infected cell culture. A simultaneous
elevation of the IC50 value was observed after 10 h PI for both molecules.
bition has been already shown for other paramyxoviruses such as
respiratory syncytial virus and human parainfluenza type 3 (De
Clercq et al., 1991; Shigeta et al., 1992; Leyssen et al., 2005), but
it has never been documented for other species of the Morbillivirus
genus.
The existence of different viral targets has been documented for
RBV, such as RNA polymerization and RNA capping enzymes for
dengue virus, vaccinia virus and hepatitis C virus (Goswami
et al., 1979; Patterson and Fernandez-Larsson, 1990; Benarroch
et al., 2004). Using a virus yield assay, we observed that both mol-
ecules were reducing in vitro CDV growth and RNA synthesis in a
time- and concentration-dependent manner. Furthermore, the re-
duced liberation of extracellular virus in the supernatants was con-
stantly associated with decreased RNA detection, proving that the
inhibition of the viral infectivity was caused by an impaired RNA
synthesis. To identify the phase of the CDV life cycle characterized
by the main antiviral activity of RBV and EICAR, we performed a
time of addition assay which clearly showed that both molecules
were active up to 10 h PI. From that moment on, they both had re-
duced antiviral activity which was completely abolished after 20 h
PI. The molecular mechanisms of the transcription and replication
of CDV are still rather poorly understood, but we have recently
shown that viral RNA can already be detected in infected cells at
8 h PI (Scagliarini et al., 2007). Plumet and colleagues (2005) ob-
served that during MV replication, viral transcription was the main
enzymatic activity of the RdRp starting during the first 5–6 h PI.
The results of the time of addition assay and of our previous data
on the dynamics of viral RNA accumulation seem to indicate that
in the early hours of infection viral transcription could be the pos-
343
B EICAR
10000
1000
RNA copies/µL
100
10
40 80 0 2 5 10 20
concentration µg/mL
48 H 72 H
25
sible target of RBV and EICAR during CDV in vitro replication.
Therefore, a direct effect of both molecules on RdRp needs to be
investigated. Our results indicate a major anti-CDV effect of RBV
20
EICAR IC50 values
and EICAR in the first hours of the viral replication cycle but further
15
experiments will be necessary to clarify the effect of RBV and EI-
(µg/mL)
CAR on the viral RdRp complex and to identify the site of interac-
10 tion between the compounds and the RdRp.
5 5. Conclusions
Our study brings new insights into the role of EICAR in the anti-
24 viral activity against CDV. EICAR showed a higher antiviral activity
but also a lower selectivity compared to RBV. These characteristics
are not compatible with its use in the therapy. More selective and
potent inhibitors need to be tested, but the results of this study will
be useful for a future characterization of the molecular and struc-
tural organization of the viral replication mechanism. Furthermore,
this study emphasizes the existence of relevant parallelism in the
antiviral activity of RBV and EICAR towards CDV and other mem-
bers of the Paramyxovirus genus. These analogies offer the opportu-
nity to use CDV as a model in the study of antiviral molecules
active against other Paramyxoviridae.
Conflict of interest statement
None declared.
Acknowledgements
This study was supported by Bologna University grants ‘‘Prog-
etti strategici di Ateneo” E.F. 2004. The authors would also like to
thank Dr. Giusy Cardeti, IZS Lazio e Toscana and Prof Fulvio Marsi-
lio, Università degli Studi di Teramo, for providing CDV strains.
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