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

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: DiNap; PRRSV; VR2332; antiviral therapeutics; pigs.

Scheme 1

Synthesis of (E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one (DiNap).

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, a prototype PRRSV, was evaluated at five different concentrations (0, 0.01, 0.02, 0.04, and 0.06 mM) in MARC-145 cells (A) and porcine alveolar macrophages (PAMs) (B) by measuring progeny virus 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 (C) and PAMs (D) at the same concentrations 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.

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.

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.

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.

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, (B) microscopic lung lesion scores, and (C) scoring of microscopic lesions were 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.

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 TCID₅₀/mL equivalents 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.