Evaluation of the inhibitory effects of (E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one (DiNap), a natural product Analog, on the replication of type 2 PRRSV in vitro and in vivo

Article abstract of DOI:10.3390/molecules24050887

DiNap [(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one], an analog of a natural product (the chalcone flavokawain), was synthesized and characterized in this study. Porcine reproductive and respiratory syndrome virus (PRRSV) is the most challenging threat to the swine industry worldwide. Currently, commercially available vaccines are ineffective for controlling porcine reproductive and respiratory syndrome (PRRS) in pigs. Therefore, a pharmacological intervention may represent an alternative control measure for PRRSV infection. Hence, the present study evaluated the effects of DiNap on the replication of VR2332 (a prototype strain of type 2 PRRSV). Initially, in vitro antiviral assays against VR2332 were performed in MARC-145 cells and porcine alveolar macrophages (PAMs). Following this, a pilot study was conducted in a pig model to demonstrate the effects of DiNap following VR2332 infection. DiNap inhibited VR2332 replication in both cell lines in a dose-dependent manner, and viral growth was completely suppressed at concentrations ≥0.06 mM, without significant cytotoxicity. Consistent with these findings, in the pig study, DiNap also reduced viral loads in the serum and lungs and enhanced the weight gain of pigs following VR2332 infection, as indicated by comparison of the DiNap-treated groups to the untreated control (NC) group. In addition, DiNap-treated pigs had fewer gross and microscopic lesions in their lungs than NC pigs. Notably, virus transmission was also delayed by approximately 1 week in uninfected contact pigs within the same group after treatment with DiNap. Taken together, these results suggest that DiNap has potential anti-PRRSV activity and could be useful as a prophylactic or post-exposure treatment drug to control PRRSV infection in pigs.

Full text of DOI:10.3390/molecules24050887

molecules
Article
Evaluation of the Inhibitory Effects of
(
(
E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-
naphthalen-1-yl)prop-2-en-1-one (DiNap),
a Natural Product Analog, on the Replication of Type
2
PRRSV In Vitro and In Vivo
Amina Khatun 1
,2,†
, Sun You Park , Nadeem Shabir 4 , Salik Nazki 1 , A-Rum Kang ,
3,†
1
1
1
1
1
3,
Chang-Gi Jeong , Byoung-Joo Seo , Myeon-Sik Yang , Bumseok Kim , Young Ho Seo *
1
,
and Won-Il Kim *
1
College of Veterinary Medicine, Chonbuk National University, Iksan 54596, Korea; aminak@jbnu.ac.kr (A.K.);
saliknazki@jbnu.ac.kr (S.N.); kar0913@jbnu.ac.kr (A.-R.K.); jcg0102@gmail.com (C.-G.J.);
byoungjooseo@gmail.com (B.-J.S.); 111@jbnu.ac.kr (M.-S.Y.); bskims@jbnu.ac.kr (B.K.)
Department of Pathology, Faculty of Animal Science and Veterinary Medicine,
2
Sher-e-Bangla Agricultural University, Dhaka 1207, Bangladesh
College of Pharmacy, Keimyung University, Daegu 42601, Korea; oasis4488@naver.com
Division of Animal Biotechnology, Faculty of Veterinary Sciences and Animal Husbandry,
3
4
Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Srinagar 190025, India;
nadeemshabir@jbnu.ac.kr
*
Correspondence: seoyho@kmu.ac.kr (Y.H.S.); kwi0621@jbnu.ac.kr (W.-I.K.);
Tel.: +82-53-580-6639 (Y.H.S.); +82-63-850-0958 (W.-I.K.);
Fax: +82-53-580-5164 (Y.H.S.); +82-63-850-0910 (W.-I.K.)
These authors have contributed equally to this work.
†
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: PhD, PharmD Raphaël E. Duval
Received: 16 December 2018; Accepted: 26 February 2019; Published: 3 March 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: DiNap [(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one],
an analog of a natural product (the chalcone flavokawain), was synthesized and characterized
in this study. Porcine reproductive and respiratory syndrome virus (PRRSV) is the most
challenging threat to the swine industry worldwide. Currently, commercially available vaccines are
ineffective for controlling porcine reproductive and respiratory syndrome (PRRS) in pigs. Therefore,
a pharmacological intervention may represent an alternative control measure for PRRSV infection.
Hence, the present study evaluated the effects of DiNap on the replication of VR2332 (a prototype
strain of type 2 PRRSV). Initially, in vitro antiviral assays against VR2332 were performed in
MARC-145 cells and porcine alveolar macrophages (PAMs). Following this, a pilot study was
conducted in a pig model to demonstrate the effects of DiNap following VR2332 infection. DiNap
inhibited VR2332 replication in both cell lines in a dose-dependent manner, and viral growth was
completely suppressed at concentrations
≥
0.06 mM, without significant cytotoxicity. Consistent with
these findings, in the pig study, DiNap also reduced viral loads in the serum and lungs and enhanced
the weight gain of pigs following VR2332 infection, as indicated by comparison of the DiNap-treated
groups to the untreated control (NC) group. In addition, DiNap-treated pigs had fewer gross and
microscopic lesions in their lungs than NC pigs. Notably, virus transmission was also delayed by
approximately 1 week in uninfected contact pigs within the same group after treatment with DiNap.
Taken together, these results suggest that DiNap has potential anti-PRRSV activity and could be
useful as a prophylactic or post-exposure treatment drug to control PRRSV infection in pigs.
Keywords: antiviral therapeutics; DiNap; PRRSV; VR2332; pigs
Molecules 2019, 24, 887; doi:10.3390/molecules24050887
www.mdpi.com/journal/molecules

Molecules 2019, 24, 887
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1
. Introduction
Porcine reproductive and respiratory syndrome (PRRS) is the most economically important
infectious disease for the swine industry worldwide. PRRS is characterized by reproductive failure
in sows and respiratory disease in pigs of all ages [ ]. The annual loss associated with PRRS is
approximately 664 million USD, as estimated by swine producers in the United States [ ]. PRRS virus
PRRSV) is a member of the Arteriviridae virus family in the order Nidovirales, along with equine arteritis
virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus
SHFV) [ ]. PRRSV is a small, enveloped virus with a single-stranded, nonsegmented, positive-sense
RNA genome that is approximately 15 kb in length [ ]. The genome of PRRSV encodes at least ten
open reading frames (ORFs) called ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5a, ORF5, ORF6,
and ORF7 [ ]. PRRSVs used to be grouped into European (type 1) and North American (type 2)
1,2
3
(
(
4,5
5,6
7,8
genotypes, but currently classified as two species, betaarterivirus suid 1 and betaarterivirus suid 2,
respectively. There is substantial genetic and antigenic variation among PRRSV strains, even within
the same virus species [9,10]. Since the first emergence of PRRS more than two decades ago, numerous
efforts have been made to develop an effective control measure for this challenging disease. Currently,
vaccination is the primary control method, and is widely used to control PRRS in pigs. However,
the use of commercial vaccines with either modified live virus (MLV) or killed virus is inadequate
to control PRRSV infection [11] because of the highly divergent PRRSV strains in the field. Antiviral
therapeutics could be an alternative or additional control measure to combat these viral infections when
a successful vaccine that matches the circulating virus is not available. Therefore, pharmacological
intervention might be a suitable and useful control strategy for PRRS. Many previous studies reported
that a variety of natural compounds had been proven to exert antiviral activity against a number of
viruses, such as equine herpesvirus 1 (EHV-1) [12], hepatitis B virus [13], human cytomegalovirus
(
HCMV) [14], and respiratory infections including the common cold [15] and PRRSV [16
However, no such effective drugs are commercially available to prevent PRRSV infection. Chalcones
1,3-diaryl-2-propen-1-ones), which constitute a key class of natural products belonging to the Flavonoid
family [30 33], were previously reported to have a wide range of biological activities, such as antiviral,
antibacterial, antifungal, immunomodulatory, anti-inflammatory, antitumor, insect antifeedant,
and antimutagenic activities [31 38]. Thus, the present study aimed to evaluate the antiviral effects
–29].
(
–
–
of DiNap [(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one], a synthetic
analog of the chalcone flavokawain, on PRRSV replication in cell culture systems. A study was then
carried out in a pig model to demonstrate the inhibitory effects of DiNap on PRRSV infection.
2
. Results
2
.1. Chemistry
The synthesis of the flavokawain analog DiNap is presented in Scheme 1, which began with the
preparation 2-hydroxy-4,6-dimethoxyacetophenone ( ). Compound was quantitatively obtained
from the reaction of 2,4,6-trihydroacetophenone ( ) with dimethyl sulfate in the presence of potassium
carbonate in acetone. The corresponding acetophenone was then subjected to the Claisen–Schmidt
condensation reaction with 1-naphthaldehyde ( ) in the presence of potassium hydroxide in methanol
to provide flavokawain analogs (DiNap) with a yield of 32%.
2
2
1
2
3

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Scheme 1. Synthesis of (E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one
DiNap).
(
2
.2. In Vitro Evaluation of the Effects of DiNap on PRRSV Replication
The antiviral effect of DiNap on PRRSV replication was evaluated in MARC-145 cells and
porcine alveolar macrophages (PAMs). DiNap reduced VR2332 replication in both cell lines in
a dose-dependent manner (Figure 1). In MARC-145 cells, VR2332 replication was decreased by
approximately 10- or 1000-fold in the presence of DiNap at concentrations of 0.02 or 0.04 mM,
respectively. Viral growth was completely suppressed at a 0.06 mM concentration of DiNap, while
the lowest concentration (0.01 mM) did not exert a significant effect on viral replication (Figure 1A).
In addition, DiNap did not show significant toxicity in MARC-145 cells, even at a concentration
of 0.06 mM at up to 48 h post treatment (hpt) (Figure 1C). Consistent with the results obtained in
MARC-145 cells, in PAMs, DiNap also reduced VR2332 replication by approximately 10- or 100-fold
in the presence of the 0.02 or 0.04 mM concentrations; the highest concentration (0.06 mM) caused
the complete suppression of VR2332 replication, while 0.01 mM DiNap was not effective (Figure 1B).
Similarly, DiNap did not cause significant toxicity in PAMs, even at concentrations of 0.06 mM at up to
4
8 hpt (Figure 1D).
Figure 1. Evaluation of the antiviral effect of DiNap on Porcine reproductive and respiratory syndrome
virus (PRRSV) replication in cells. The antiviral activity of DiNap against the replication of VR2332,

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a prototype PRRSV, was evaluated at five different concentrations (0, 0.01, 0.02, 0.04, and 0.06 mM)
in MARC-145 cells ( ) and porcine alveolar macrophages (PAMs) ( ) by measuring progeny virus
A
B
production over time. Both types of cells were prepared in 6-well cell culture plates, and the cell
monolayers were pretreated for 2 h before infection with growth medium containing DiNap. Then,
the cells were inoculated with VR2332 at an MOI of 0.01 and incubated for 1 additional hour.
The cell monolayers were replenished with growth medium containing the same concentrations
of DiNap and incubated for 4 additional days under the same culture conditions. An MTT
[
3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was also conducted on both cell
lines to assess the cytotoxicity of DiNap in MARC-145 cells ( ) and PAMs ( ) at the same concentrations
C
D
described above. The cytotoxicity data are expressed as the relative cell viability. Asterisks indicate
significant differences (* p < 0.05, ** p < 0.001) in viral replication between groups.
2
.3. Inhibitory Effect of DiNap on PRRSV in Experimentally Infected Pigs
2
.3.1. Serum Viremia
The viral loads in the serum samples were quantified by real-time reverse transcription-polymerase
®
chain reaction (qRT-PCR) (TaqMan ), and the results are summarized in Figure 2. One pig (1, 4 or 7) in
each group was challenged with VR2332 and maintained in the group with the other two unchallenged
pigs to observe the effects of DiNap on virus transmission between challenged and unchallenged pigs.
In group 1 (NC), the pig (1) challenged with VR2332 had the highest viral load, with virus titers of
1.39
1
2
05.55 TCID /mL at 7 days post challenge (dpc), which gradually decreased to 10
TCID /mL at
50
5
0
8 dpc. Similarly, the two unchallenged pigs (2 and 3) in the same group also showed peak viral titers of
3.83
4.64
1.23
2.01
1
0
and 10
TCID /mL at 7 dpc, which also gradually decreased to 10
and 10
TCID /mL,
50
50
respectively, at 28 dpc (Figure 2A).
In group 2 (DiNap at 0.04 mM/kg body weight), the pig (4) challenged with VR2332 had a peak
viral titer of 104 TCID /mL at 7 dpc, which was more than 10-fold lower than the titers found in the
.18
50
0.93
pig (1) challenged with the virus in group 1; the viral titer gradually decreased to 10
TCID /mL at
50
2
8 dpc. Among the two unchallenged pigs in the same group, one pig (5) showed a peak virus titer of
3.25
1
0
TCID /mL at 7 dpc, which was also lower than the titers in both unchallenged pigs (2 and 3)
50
1.39
in group 1, and the titer gradually declined to 10
TCID /mL at 28 dpc. The other unchallenged
50
pig (6) did not show viremia at up to 7 dpc, although the virus was detected at 14 dpc, with a titer of
3.14
2.19
1
0
TCID /mL, which slowly decreased to 10
TCID /mL at 28 dpc (Figure 2A).
50
50
Consistent with these results, in group 3 (DiNap at 0.2 mM/kg body weight), the pig (7) challenged
with VR2332 had peak viremia with a virus titer of 104.08 TCID /mL at 7 dpc, which was more than
50
1
0-fold lower than the titer found in the pig (1) challenged with virus in group 1, and the virus titer
gradually decreased to 101 TCID /mL at 28 dpc. Neither unchallenged pig (8 and 9) in the same
.02
50
group displayed viremia at up to 7 dpc, although the virus was detected in both of the pigs at 14 dpc,
with titers of 102 and 10
respectively, at 28 dpc (Figure 2A).
In addition, the average virus titers were measured in the pigs in each group. The pigs in
.17
2.92
TCID /mL, which slowly declined to 10
1.24
and 10
1.51
TCID /mL,
50
50
4.67
group 1 (NC) exhibited an average peak viremia titer of 10
TCID /mL at 7 dpc, which gradually
50
decreased to 101 TCID /mL at 28 dpc. In contrast, the pigs in group 2 (DiNap at 0.04 mM) and
.54
50
2.47
1.36
3
(DiNap at 0.2 mM) had average titers of 10
TCID /mL and 10
TCID /mL, respectively,
50
5
0
at 7 dpc, which were approximately 100-fold (p < 0.05) and 1000-fold (p < 0.001) lower than the average
titer observed in the pigs in group 1 (NC) (Figure 2B). These results suggest that DiNap has significant
antiviral effects against PRRSV, decreasing viral transmission and replication in pigs.

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Figure 2. Serum viremia was measured in pig serum from each group at 0 (before challenge), 7, 14,
®
and 28 dpc by real-time reverse transcription-polymerase chain reaction (qRT-PCR) (TaqMan ). Viral
RNA was extracted from serum samples, and the viral loads were quantified by qRT-PCR. The level of
viremia was calculated and expressed as a TCID50/mL equivalent based on the standard curve of the
cycle threshold (Ct) number plotted against the known virus titer of VR2332. The measured virus titers
in the serum samples are presented for individual animals (A) and as an average for each group (B).
Asterisks indicate significant differences (* p < 0.05) in virus titers between groups. Note: v indicates
the pig challenged with virus in each group.
2
.3.2. Anti-PRRSV IgG Response
The PRRSV-specific antibody (IgG) response was measured in serum samples by enzyme-linked
immunosorbent assay (ELISA) based on the nucleocapsid (N) protein. As summarized in Figure 3,
all pigs in each group were seronegative at up to 7 dpc. Following infection, all 3 pigs (1 to 3) in group
1
2
(NC) became seropositive at 14 dpc, and the S/P (sample-to-positive) ratio gradually increased up to
8 dpc. Similarly, in group 2, two pigs [virus challenged (4) and unchallenged (5)] became seropositive
at 14 dpc. The other unchallenged pig (6) in the same group was seronegative at up to 14 dpc and
became seropositive at 21 dpc. In group 3, the challenged pig (7) became seropositive at 14 dpc, similar
to the challenged pig (1) in group 1. However, the two other unchallenged pigs (8 and 9) in the same
group were seronegative at up to 14 dpc and became seropositive at 21 dpc. There were no differences
in the antibody (IgG) titers found among the groups (Figure 3). These results suggested that DiNap
delayed viral transmission among pigs infected with PRRSV.

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Figure 3. Anti-PRRSV antibody (IgG) responses measured by enzyme-linked immunosorbent assay
ELISA) in the pigs in each group. PRRSV-specific antibodies (IgG) were detected in the serum samples
by using a commercially available ELISA kit (IDEXX PRRS X3 Ab Test). An S/P ratio (ratio between
the net optical density of the test samples and the net optical density of the positive control) 0.4 was
considered positive for the PRRSV antibody. The results are expressed as the S/P ratio of the samples,
with S/P 0.4 considered positive for the PRRSV antibody (IgG). Note: v indicates the pig challenged
(
≥
≥
with virus in each group.
2
.3.3. Average Daily Weight Gain
The effect of DiNap on the growth rate of the pigs was evaluated over the course of PRRSV
infection. The average daily weight gain (ADWG) was measured for all pigs in each group up to
8 dpc. DiNap significantly enhanced the ADWG for both treatment groups (groups 2 and 3) compared
with that of the pigs in group 1 (NC) (Figure 4).
2

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Figure 4. Assessment of average daily weight gain (ADWG). ADWG was calculated for the pigs in
each group at 28 days and is expressed in kg/day. Asterisks indicate significant differences (* p < 0.05)
in the ADWG of the pigs between groups.
2
.3.4. Lung Pathology and Residual Viral Loads in Lung Tissues
All pigs in the untreated control group (group 1) exhibited higher lung lesion scores (gross and
microscopic) than those in group 2 and group 3 (Figure 5).
Figure 5. Lung pathology. Scoring of lung lesions was performed for all pigs in each group.
Lung scores were plotted as the mean values of lesions scored in the five lobes of the lung. (
A) Gross
lung lesion scores, ( ) microscopic lung lesion scores, and ( ) scoring of microscopic lesions were
B
C
recorded according to a 4-point scale from 0 to 3, with 0 indicating no lesion, 1 indicating mild
interstitial pneumonia, 2 indicating moderate/multifocal interstitial pneumonia, and 3 indicating
severe intense/diffuse interstitial pneumonia. Asterisks indicate significant differences (* p < 0.05) in
pig lung lesions between groups.

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The residual viral loads were also measured in the lung tissues collected from the pigs in each
group. DiNap appeared to reduce the viral loads observed in the lung tissues of the pigs in each
treatment group (groups 2 and 3) compared to those in group 1 (NC). The pigs in groups 2 and 3
exhibited average virus titers of 103 TCID /mL and 10
.28
2.68
TCID /mL, respectively, which were
50
50
4.65
approximately 10- and 100-fold lower than the titer of 10
, the pig (1) challenged with VR2332 had a virus titer of 10
and the unchallenged pigs (2 and 3) in the same group exhibited virus titers of 10
TCID /mL, respectively. However, in group 2, the pig (4) challenged with the virus had a virus titer
of 102 TCID /mL, which was approximately 100-fold lower than the titer found in the challenged
pig (1) in group 1. The two unchallenged pigs (5 and 6) in the same group had titers of 10
TCID /mL observed in group 1. In group
50
4
.84
1
TCID /mL in its lung tissue,
50
4.94
4.17
and 10
5
0
.10
50
3.51
and
4.24
10
TCID /mL, respectively, which were also lower than or similar to the titers found in both
5
0
unchallenged pigs (2 and 3) in group 1. In group 3, the pig (7) that was challenged with the virus
exhibited a virus titer of 101 TCID /mL in its lung tissues, which was also more than 100-fold
.98
50
lower than the titer found in the pig (1) that was challenged by the virus in group 1. Moreover,
both unchallenged pigs (8 and 9) in the same group also had lower virus titers of 102 and 10
.98
3.11
TCID /mL, respectively, in their lung tissues than the titers found in both unchallenged pigs (2 and 3)
50
in group 1 (Figure 6).
Figure 6. Quantification of residual viral loads in lung tissues collected from pigs in each group at
28 dpc. Viral RNA was extracted from the lung, and viral loads in the lung tissues were quantified by
qRT-PCR. The viral loads in lung tissues were measured and expressed as TCID /mL equivalents
5
0
based on the standard curve of the Ct number plotted against the known virus titer of VR2332. Asterisks
indicate significant differences (* p < 0.05) in virus titers between groups.
3
. Discussion
The chalcone flavokawain was found to exhibit promising anticancer and anti-inflammatory
properties in previous studies [39 40]. DiNap, a synthetic analog of the chalcone flavokawain,
,
was evaluated in this study for its potential antiviral activity against PRRSV infection in cell culture
systems and in a pig model. Chemically, DiNap is a synthetic analog of flavokawain, containing

Molecules 2019, 24, 887
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a chalcone scaffold of 1,3-diaryl-2-propen-1-one. DiNap has a 2-hydroxy-4,6-dimethoxybenzene
moiety at the C1 position of 2-propene-1-one, which is a common structural feature of the naturally
occurring compounds flavokawain A, B, and C. DiNap also contains a naphthalenyl moiety at the
C3 position of 2-propene-1-one, which has been found in several antiviral agents [41,42]. In addition,
chalcones, the main pharmacophore of DiNap, have been reported to show a wide spectrum of
biological activities, including antiviral [43], antioxidative [44], and anti-inflammatory activities [45].
These previous studies are further supported by the results presented in the current study. DiNap
effectively suppressed VR2332 replication in MARC-145 cells and PAMs at concentrations ranging
from 0.02 to 0.06 mM, without showing significant cytotoxicity, even at up to 48 hpt. It is worth noting
that the highest concentration of DiNap (0.06 mM) used in this study completely suppressed VR2332
replication in both cell lines (Figure 1). Although approximately 20% cell death was observed when
DiNap was added at 0.06 mM, no significant side effects or toxicity were observed after treatment
in pigs.
In the pig experiment, we demonstrated that DiNap exerted its potential inhibitory effects against
PRRSV infectivity in a dose-dependent manner. Compared to the pigs in the untreated control group
(
group 1), the pigs treated with DiNap in group 2 and group 3 had reduced viral loads in the serum and
lungs and increased weight gain (Figures 2, 4 and 6). Consistent with these findings, the pigs treated
with DiNap also had fewer lesions (gross and microscopic) in their lungs than the control pigs (Figure 5),
suggesting that DiNap exerted significant effects on PRRSV infection. More importantly, two injections
of DiNap were able to delay virus transmission by approximately 1 week with direct contact between
the infected (virus challenged) and uninfected (not virus challenged) pigs in both treatment groups
(groups 2 and 3). However, the virus was easily transmitted between the infected and uninfected pigs
in the untreated control group (group 1), indicating that DiNap could be useful as a prophylactic or
postexposure treatment agent in cases of PRRSV infection. Because DiNap was produced in limited
quantities on a laboratory scale, the antiviral effect of DiNap was only preliminarily evaluated with
a small number of pigs in the current study. Therefore, more detailed animal experiments are needed
to determine how long the antiviral effect of DiNap lasts in the future.
Taken together, these results demonstrated that DiNap might be useful as a potential prophylactic
and postexposure anti-PRRSV drug for the treatment of PRRSV-infected pigs, although the efficacy and
safety of long-term use of higher concentrations of DiNap remains to be confirmed in future experiments.
4
4
4
. Materials and Methods
.1. Synthesis of DiNap, an Analog of a Natural Product
.1.1. General Methods
All reagents and solvents were purchased from Sigma-Aldrich (Milwaukee, WI, USA) and Alfa
Aesar (Ward Hill, MA, USA). All experiments involving moisture-sensitive compounds were carried
out in an argon atmosphere. Analytical thin-layer chromatography was performed on precoated
silica gel F254 thin layer chromatography (TLC) plates (E, Merck, Kenilworth, NJ, USA) with
visualization under UV light. The final products were purified by preparative medium-pressure
liquid chromatography (MPLC) (Biotage Isolera One instrument) on a silica column (Biotage SNAP
HP-Sil) spectrometer (Biotage, Uppsala, Sweden). Nuclear magnetic resonance (NMR) analyses were
1
13
performed using a Bruker 400 (400 MHz for H; 100 MHz for C) spectrometer (Bruker, Rheinstetten,
Germany). The chemical shifts are reported in parts per million ( ). The deuterium lock signal of
δ
the sample solvent was used as a reference, and the coupling constants (J) are given in hertz (Hz).
The splitting pattern abbreviations are as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd,
doublet of doublets; and m, multiplet. The purity of all tested compounds was confirmed to be higher
than 95% by analytical high-performance liquid chromatography (HPLC) analysis performed with
a dual-pump Shimadzu LC-6AD system equipped with a VP-ODS C18 column (4.6 mm
µm, Shimadzu).
×
250 mm,
5

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4
.1.2. 1-(2-Hydroxy-4,6-dimethoxyphenyl)ethanone (2)
0
0
0
A mixture of 2 ,4 ,6 -trihydroacetophenone hydrate (2.0 g, 11.89 mmol), potassium carbonate
(
3.62 g, 26.17 mmol) and dimethylsulfate (2.32 mL, 24.38 mmol) in acetone (30 mL) was stirred under
◦
argon at 66 C for 2 h. The resulting mixture was cooled to room temperature, filtered, and washed
with acetone. The filtrate was extracted with ethyl acetate. The organic layer was washed three times
with saturated NaHCO solution, dried over Na SO , and concentrated under reduced pressure to
3
2
4
1
produce compound
2 with 100% yield (R = 0.34 (1:9 ethyl acetate: hexane); H NMR (400 MHz, CDCl )
f
3
δ 14.06 (s, 1H), 6.05 (d, J = 2.0 Hz, 1H), 5.81 (d, J = 2.4 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 2.61 (s, 3H)).
4
.1.3. (E)-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one (DiNap)
DiNap was synthesized following the procedure reported in the literature with slight
modifications [46]. Briefly, a mixture of compound
2
(0.2 g, 1.02 mmol), 1-naphthaldehyde (0.14 mL,
◦
1.02 mmol) and potassium hydroxide (1 g) in methanol (20 mL) was stirred at 50 C for 3 days.
The mixture was extracted with ethyl acetate. The organic layer was washed three times with water,
dried over Na SO , concentrated under reduced pressure, and purified by MPLC to produce DiNap
2
4
1
with 32% yield (R = 0.27 (1:9 ethyl acetate: hexane); H NMR (400 MHz, CDCl )
δ
14.40 (s, 1H),
.61 (d, J = 15.6 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 15.2 Hz, 1H), 7.90–7.87 (m, 2H), 7.83
d, J = 7.2 Hz, 1H), 7.60–7.49 (m, 3H), 6.13 (d, J = 2.0, 1H), 5.96 (d, J = 2.4 Hz, 1H), 3.89 (s, 3H), 3.823
s, 3H); 13C NMR (100 MHz, CDCl3)
192.8, 168.7, 166.6, 162.8, 139.3, 134.0, 133.2, 132.0, 130.6, 130.4,
29.0, 127.0, 126.4, 125.7, 125.4, 123.9, 106.6, 94.1, 91.5, 57.4, 56.1).
After synthesis, DiNap was dissolved in dimethyl sulfoxide (DMSO) (Sigma, Aldrich, St. Louis,
f
3
8
(
(
δ
1
MO, USA) at a stock concentration of 20 mM, sterile-filtered using a 0.20-
Germany), aliquoted and stored at 4 C until use.
µm syringe filter (Corning,
◦
4
.2. Viruses and Cells
VR2332, a prototype strain of type 2 PRRSV, was used in the present study. MARC-145, an African
Green Monkey Kidney cell line (highly permissive to PRRSV) [47], and PAMs (the primary target
cell for PRRSV replication in pigs) [48 49] were used for the in vitro experiments. PAMs were
collected from 6-week-old, PRRSV-free pigs by bronchoalveolar lavage, as described previously [50 51].
,
,
MARC-145 cells were used for virus propagation, while the antiviral assays were performed in both
cell lines (MARC-145 and PAMs). Both cell lines were maintained in RPMI-1640 medium (Gibco,
Invitrogen, Carlsbad, CA, USA) supplemented with heat-inactivated 10% fetal bovine serum (FBS,
Gibco, Invitrogen), 2 mM L-glutamine, and 100x antibiotic-antimycotic solution (Anti-anti, Invitrogen;
®
1
x solution contains 100 IU/mL penicillin, 100
µ
g/mL streptomycin, and 0.25
µ
g/mL Fungizone
◦
(amphotericin B)) (named “growth medium”) at 37 C in a humidified 5% CO atmosphere.
2
4
.3. Antiviral Effect of DiNap Against PRRSV Replication in Cells
MARC-145 cells and PAMs were prepared in 6-well cell culture plates (BD, Falcon) 24 to 48 h before
the start of the experiment. Following this, the cell monolayers were pretreated for 2 h before infection
with growth medium containing DiNap at five different concentrations (0, 0.01, 0.02, 0.04, and 0.06 mM).
After the pretreatment incubation, the cell monolayers were washed with growth medium, inoculated
with VR2332 at a multiplicity of infection (MOI) of 0.01 and incubated for 1 h. After the final 1-h
incubation, the viral inoculum was discarded, and the cell monolayers were replenished with growth
medium containing the same concentrations of DiNap as a treatment and incubated for 4 more days
under the same culture conditions. The cell culture supernatants were collected every 24 h, centrifuged,
◦
and stored at
−
80 C until analysis. The progeny virus titers were measured using a microtitration
infectivity assay [52]; the virus titration assay was described briefly in a previous study [53]. The virus
titers were calculated based on the cytopathic effect (CPE) and are expressed as TCID /mL [54].
50

Molecules 2019, 24, 887
11 of 16
4
.4. MTT Assay
The cytotoxic effect of DiNap was assessed in MARC-145 cells and PAMs using an MTT
3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT, Sigma-Aldrich, St. Louis, MO,
USA) assay, as described previously [55 56]. In brief, MARC-145 cells and PAMs were prepared in
6-well cell culture plates (BD, Falcon) and treated with growth medium containing DiNap (0, 0.01,
.02, 0.04, and 0.06 mM) for 24 and 48 h. After incubation, the cell supernatants were discarded,
L of freshly made MTT (5 mg/mL) solution was added along with growth medium to each
[
,
9
0
and 20
µ
well. Then, the cells were incubated for another 3 h under the same conditions. After incubation,
◦
the MTT solutions were removed, and the plates were dried at 65 C overnight. Then, 200
µL of
®
dimethyl sulfoxide (DMSO; Hybri-Max , Sigma-Aldrich) was added to each well to dissolve the
formazan crystals. The plates were further incubated at 37 C for 5 to 10 min to dissolve any air
◦
bubbles before the MTT signal was measured using an ELISA microplate reader at an absorbance of
5
40 nm.
4
.5. Animal Experiment and Samples
In total, 9 three-week-old PRRSV-negative pigs were purchased. On their arrival at the facility,
the pigs were randomly housed in three groups (3 pigs per group), designated group 1 (pigs 1 to 3),
group 2 (pigs 4 to 6), and group 3 (pigs 7 to 9) and allowed to acclimatize for 3 days. Then, qRT-PCR
(
TaqMan) and ELISA were used to confirm that the pigs were negative for PRRSV. On day 0, one pig
(
1, 4, and 7 in groups 1, 2,and 3, respectively) in each group was challenged with VR2332 at a titer
3
of 10 /mL TCID50 (2 mL/pig) through the intranasal (IN) route, and the challenged pigs were then
maintained in their groups to observe the transmission of the virus to the unchallenged contact pigs.
On the same day, all 3 pigs in group 2 and group 3 were injected with 0.32 mM (0.04 mM/kg body
weight) and 1.6 mM (0.2 mM/kg body weight) DiNap prepared in 2 mL of DMSO behind the base of
each ear intramuscularly (IM), respectively. All 3 pigs in group 1 constituted the nontreated challenged
control (NC) group. The treatment with DiNap was repeated with the same concentrations at 3 dpc.
After this, the pigs were observed daily for clinical signs up to 28 dpc. The pigs were weighed
at 0 (before challenge) and 28 dpc. Serum samples were collected at 0 (before challenge), 7, 14, 21,
and 28 dpc for virological and serological assays. At the end of 28 dpc, all pigs were euthanized, and the
results of pathological examinations, including the presence of gross lung lesions, were recorded by
an expert pathologist based on the standard scoring system, as described previously [57]. In addition,
lung tissue samples were collected in 10% neutral buffered formalin for the examination of microscopic
◦
changes. Furthermore, additional lung tissue samples were collected and stored at
−
80 C until
analysis. Viral loads were quantified in the serum and lung tissue samples. The animal experiment
protocols were approved by the Chonbuk National University Institutional Animal Care and Use
Committee (approval number: 2012-0025).
4
.5.1. Serum ELISA
PRRSV-specific antibodies (IgG) were detected in the serum samples from each group by using
a commercially available ELISA kit (IDEXX PRRS X3 Ab Test, IDEXX Laboratories, AG, Berne,
Switzerland) following the instructions supplied by the manufacturer. An S/P ratio (ratio between the
net optical density of the test samples and the net optical density of the positive control)
considered positive for the PRRSV antibody.
≥
0.4 was
4
.5.2. Lung Tissue Scoring and Pathology
At the end of 28 dpc, the lungs of the euthanized pigs were scored for gross lesions immediately
after necropsy [57]. The microscopic lesions were also scored in lung tissue sections by staining
with hematoxylin and eosin (H & E). The scoring of the microscopic lesions was performed
according to a scale from 0 to 3 depending on the degree of inflammation (especially the presence

Molecules 2019, 24, 887
12 of 16
of interstitial pneumonia), where 0 indicated no lesions, 1 indicated mild interstitial pneumonia,
indicated moderate/multifocal interstitial pneumonia, and 3 indicated severe intense/diffuse
interstitial pneumonia.
2
4
.5.3. Quantification of Viral Loads in Serum and Lung Tissue
Viral RNA was extracted from the serum and lung tissue samples using the MagMAX
™ Viral
RNA Isolation Kit (Ambion, Applied Biosystems, Life Technologies, Inc., Carlsbad, CA, USA)
and a total RNA extraction kit (Hybrid-RTM, GeneAll, Seoul, Korea), respectively, according
to the manufacturer’s instructions. The viral loads in the serum and tissues were quantified by
®
real-time reverse transcription-polymerase chain reaction (qRT-PCR) using a TaqMan assay based
on a previous study [58]. The primer and probe sequences were as follows: forward primer
TGTCAGATTCAGGGAGRATAAGTTAC; probe 6-FAM TGTGGAGTTYAGTYTGCC; and reverse
primer ATCARGCGCACAGTRTGATGC. A one-step quantitative real-time RT-PCR (qRT-PCR) kit
(
AgPath-ID
CA, USA) was used to measure the viral loads in the serum and lung tissues. Polymerase chain reaction
PCR) amplification was performed with a 7500 Fast Real-time PCR system (Applied Biosystems,
Foster City, CA, USA) according to the manufacturer’s guidelines. One-step qRT-PCR was performed
™ One-Step RT-PCR, Ambion, Applied Biosystems, Life Technologies, Inc., Carlsbad,
(
in a total of 25
forward and reverse primer (20 pmol, at a final concentration of 0.8 pmol), 0.2
probes (25 pmol, at a final concentration of 1 pmol), 0.5 L of RNAse inhibitor (40 U/
Thermo Fisher Scientific Inc., Germany), 1 L of 25X RT-PCR enzyme mix, and 4.8
µ
L containing 5
µ
L of template RNA, 12.5
µ
L of 2
×
RT-PCR buffer, 0.5
µ
L of each
®
µ
L of the TaqMan
µ
µ
L; RiboLockTM,
µ
µ
L of nuclease-free
◦
water. The cycling conditions were as follows: (a) Reverse transcription for 10 min at 45 C; (b) a 10-min
◦
◦
◦
activation step at 95 C; and (c) 40 cycles of 15 s at 95 C and 45 s at 60 C. Samples with a threshold
cycle (C ) of 35 cycles or fewer were considered positive. A standard curve was generated from known
virus titers and used to calculate the amount of PRRSV in each sample by converting the C value to
the TCID /mL equivalent.
t
t
50
4
.6. Data Analysis
Repeated measures analysis of variance (ANOVA) was used to analyze the significance of
differences among the treatment groups for the in vitro evaluation of the effect of DiNap on
PRRSV replication in cells and for the serum viremia and serum ELISA (anti-PRRSV IgG) results.
The Mann–Whitney U test (a nonparametric t test) was used to analyze the significance of differences
among groups for ADWG, lung lesions (gross and microscopic) and viral loads in lung tissues. p < 0.05
indicated a statistically significant difference. Graph Pad Prism 5.0.2 (GraphPad Software, Inc., CA,
USA) was used to produce the graphs, while statistical analysis was performed using SPSS Advanced
Statistics 17.0 software (SPSS, Inc., Chicago, IL, USA).
Author Contributions: A.K. and S.Y.P. designed and performed all the experiments, analyzed and interpreted all
the results, and drafted the manuscript. N.S., S.N., A.-R.K., C.-G.J., and B.-J.S. contributed to the experiments
and the data analysis. M.-S.Y. and B.K. were involved in animal experiments, including clinical and postmortem
examinations. Y.H.S. and W.-I.K. conceived the study, developed the principal ideas, coordinated the work
described, and critically reviewed the drafted manuscript. All authors read and approved the final manuscript.
Funding: This study was supported by the Technology Development Program for Bioindustry (Project No. 116102-3)
of the Ministry for Food, Agriculture, Forestry and Fisheries of the Republic of Korea.
Acknowledgments: The authors are happy to acknowledge the undergraduate students and laboratory
technicians from the College of Veterinary Medicine and Veterinary Diagnostic Center (VDC), respectively,
Chonbuk National University for their assistance during the animal experiment.
Conflicts of Interest: The authors declared that there are no conflicts of interest.

Molecules 2019, 24, 887
13 of 16
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Sample Availability: Not available.
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