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. 2018 Feb 28;13(2):e0193309.
doi: 10.1371/journal.pone.0193309. eCollection 2018.

Inhibition of proanthocyanidin A2 on porcine reproductive and respiratory syndrome virus replication in vitro

Mingxin Zhang  1 , Qianqian Wu  1 , Yao Chen  2 , Mubing Duan  3 , Ge Tian  1 , Xianbo Deng  1 , Yankuo Sun  2 , Tong Zhou  1 , Guihong Zhang  2 , Weisan Chen  3 , Jianxin Chen  1
Affiliations

Inhibition of proanthocyanidin A2 on porcine reproductive and respiratory syndrome virus replication in vitro

Mingxin Zhang et al. PLoS One. .

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely prevalent and endemic swine pathogen that causes significant economic losses for the global pig industry annually. Currently, the most prevalent strategy for PRRSV control remains the prevention of virus transmission, with highly effective therapeutic agents and vaccines still lacking. Proanthocyanidin A2 (PA2) belongs to the family of tea polyphenols, which have been reported to exhibit a range of biological activities including anti-oxidative, cardio-protective, anti-tumoural, anti-bacterial, anti-viral, and anti-inflammatory effects in vitro as well as in vivo. Here, we demonstrate that PA2 exhibits potent anti-viral activity against PRRSV infection in Marc-145 cells. Similar inhibitory effects were also found in porcine alveolar macrophages, the primary target cell type of PRRSV infection in pigs in vivo. For traditional type II PRRSV CH-1a strain and high pathogenic GD-XH strain and GD-HD strain, PA2 exhibited broad-spectrum and comparable inhibitory activities in vitro with EC50 ranging from 2.2 to 3.2 μg/ml. Treatment of PRRSV-infected Marc-145 cells with PA2 significantly inhibited viral RNA synthesis, viral protein expression and progeny virus production in a dose-dependent manner. In addition, PA2 treatment reduced gene expressions of cytokines (TNF-α, IFN-α, IL-1β and IL-6) induced by PRRSV infection in PAMs. Mechanistically, PA2 inhibited PRRSV replication by targeting multiple pathways including blockade of viral entry and progeny virus release. Altogether, our findings suggest that PA2 has the potential to serve as a novel prophylactic and therapeutic strategies against PRRSV infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The anti-PRRSV activity and cellular toxicity of PA2 in Marc-145 cell cultures.
(A) Chemical structures of proanthocyanidin A2 (PA2). (B) Cellular toxicity was examined in Marc-145 cells and PAMs using Cell Counting Kit-8 and was expressed as relative cell viability by comparing with the viable cells in the absence of compound (set up as 100%). (C and D) Antiviral activity of PA2 against the PRRSV CH-1a strain and high pathogenic GD-XH strain in Marc-145 cells was examined using IFA. Data shown in C are one representative of three independent experiments corresponding to D. Cells grown in 96-well plates were infected with PRRSV (0.05 MOI) for 2 h at 37°C and then cultured in fresh medium containing various concentrations of PA2. IFA for the N protein of PRRSV was performed at 48 hpi. Upper panels in C: GD-XH using Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (green). Lower panels in C: CH-1a using Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (red). Scale bar: 100 μm. Results shown in D are the mean values of relative fluorescence intensity of anti-N protein from three independent IFA experiments, and DMSO-treated control (0 μg/ml PA2) was set as 100%. Software Image J was used to analyze fluorescence intensity of IFA images. Statistical significances are denoted by *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 2
Fig 2. Confirmation of anti-PRRSV activity of PA2 in Marc-145 cell cultures.
Cells grown in 6-well plates were infected with PRRSV GD-XH (0.05 MOI) for 2 h at 37°C and then cultured in fresh medium containing various concentrations of PA2. At 48 h (A, B, D and E) or indicated time-points (C, F and G) post infection, the samples were subjected to viral titer titration, or RT-PCR or Western blotting analysis. (A) The PRRSV titer was determined after treatment with PA2 for 48 h using the end point dilution assay and expressed as log10 TCID50/ml. (B and C) Relative PRRSV NSP9 mRNA level was analyzed using real-time RT-PCR at 48 h (B) or indicated time-points (C) after treatment with PA2. Expression of GAPDH was shown as the internal loading control, and DMSO-treated sample (0 μg/ml PA2) at 48 h was used as the reference control (set as 1). Results shown in A, B and C are the mean values from three independent experiments, and error bars represent standard deviations. (D and E) Expression of viral N protein in cells treated with various concentrations of PA2 for 48 h was detected by Western blotting. (F and G) Expression of viral N protein in cells treated with 40 μg/ml PA2 or 40 μg/ml ribavirin for various hours was detected by Western blotting. β-actin was used as a loading control. Results shown in E and G are normalized N protein levels based on the optical densities (OD) of bands from three independent experiments. Software Image J was used to analyze band OD; Results from PA2 treated samples were compared to those from DMSO-treated control groups (0 μg/ml PA2) for 48 h. Data shown in D and F are one representative of three independent experiments corresponding to E and G, respectively. Statistical significances are denoted by *p or #p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 3
Fig 3. The anti-PRRSV activity of PA2 in PAM cultures.
PAMs grown in 96-well plates (A and B) or 6-well plates (C and D) were infected with PRRSV GD-HD (0.1 MOI) for 2 h at 37°C and then cultured in fresh medium containing various concentrations of PA2. After treatment with PA2 for 24 h, the samples were subjected to IFA, or viral titer titration and RT-PCR analysis. (A and B) Indirect immunofluorescence assay for the N protein of PRRSV was performed at 24 hpi. For staining, Alexa Fluor 568-conjugated goat anti-mouse antibody was used as the secondary antibody (red), and nuclei were stained with DAPI (blue). Scale bar: 100 μm. Results shown in B are the mean values of relative fluorescence intensity of anti-N protein from three independent IFA experiments (DMSO-treated control as 100%), and A is one representative data from B. (C) The PRRSV titer was determined after treatment with PA2 for 24 h using the end point dilution assay and expressed as log10 TCID50/ml. (D) Relative PRRSV NSP9 mRNA expression of PA2 treated groups to DMSO-treated control (0 μg/ml PA2) (set as 1) was analyzed using real-time RT-PCR at 24 h after treatment with PA2. Results shown in C and D are the mean values from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to DMSO-treated control.
Fig 4
Fig 4. Effect of PA2 treatment on PRRSV entry, replication and release.
Marc-145 cells were infected with the PRRSV GD-XH strain at 0.05 MOI. The infected cells were cultured in the presence of a series of concentrations of PA2 and collected at indicated time-points post infection for determination of the relative expression level of viral NSP9 mRNA by RT-PCR. Cellular GAPDH mRNA was used as the internal loading control and DMSO-treated sample (set as 1) was used as the reference control. (A) Different PA2 treatment schemes. Red bars represent PRRSV infection period, blue bars represent PA2 treatment period, and red vertical bars represent end of treatments and cell harvesting. (B) Viral binding was investigated by treatment A1; (C) Viral internalization was investigated by treatment A2; (D) Viral replication by treatment A3; (E) Viral release by treatment A4; and (F) PA2 pretreatment was performed by treatment A5. The data (in B, C, D, E and F) represent the mean ± standard deviation from three independent experiments. Statistical significances are denoted by *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 5
Fig 5. Effect of PA2 treatment on cytokine gene expression induced by PRRSV infection in PAMs.
PAMs grown in 6-well plates were infected with PRRSV GD-HD (0.1 MOI) for 2 h at 37°C and then cultured in fresh medium in the presence or absence of 40 μg/ml PA2. Total RNA was extracted from lysates of PAMs at 12 hpi and 24 hpi. The mRNA level of each cytokine gene was assessed by RT-PCR using cellular GAPDH mRNA as the internal loading control. Relative expression (fold change) in comparison with DMSO-treated mock-infected cells (set as 1) is shown. The data represent the mean values from three independent experiments. Statistical significances are denoted by *p < 0.05, **p < 0.01, and ***p < 0.001.
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This work was supported by the National Natural Science Foundation of China (Grant No: 31572565, 30972217), the National Key Research and Development Program of China (Grant No: 2016YFD0501300, 2017YFD0501404) and the Natural Science Foundation of Guangdong Province (Grant No: 2015A030313399), all to JC.

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