In vitro evaluation of antiviral activity of tea seed saponins against porcine reproductive and respiratory syndrome virus E Li, Na Sun, Jun-Xing Zhao, Yao-Gui Sun, Jian-Gang Huang, Hai-Min Lei, Jian-Hua Guo, Yuan-Liang Hu, Wen-Kui Wang, Hong-Quan Li Antiviral Therapy 2015; 10.3851/IMP2937 Submission date Acceptance date Publication date

21st December 2014 15th January 2015 22nd January 2015

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Publication: Antiviral Therapy; Type: Original article DOI: 10.3851/IMP2937

Original article In vitro evaluation of antiviral activity of tea seed saponins against porcine reproductive and respiratory syndrome virus E Li1†, Na Sun1†, Jun-Xing Zhao1, Yao-Gui Sun1, Jian-Gang Huang1, Hai-Min Lei2, JianHua Guo3, Yuan-Liang Hu4, Wen-Kui Wang1, Hong-Quan Li1* 1

College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, PR China 2

School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing, PR China

3

Department of Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA 4

Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, PR China *Corresponding author e-mail: [email protected] †

Equal contribution to this work

Abstract Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of major pathogens of swine. This virus cause immune suppression and other secondary infections, leading significant economic losses in the swine industry. Tea seeds saponins (TS) is a natural extract from the tea seeds with anti-cancer, anti-inflammatory activity, and antiviral activity. In this study, we demonstrated that TS possessed anti-PRRSV activity. Methods: MTT assay and Trypan blue Staining were used to evaluate the cytotoxicity and antiviral ability of TS in cell culture. Apoptosis was measured to assess the safety of TS on Marc-145 cells. Moreover, time-of-addition assay, entry inhibition assay and virucidal assay were used to assess the antiviral action of TS. The effect of TS on host cellular gene expression was analyzed by real-time PCR. Absolute quantification RT-PCR and Western blot were used to study the inhibitory effect of TS on PRRSV N gene and protein expression. Results: Our results showed that 50% cytotoxic concentrations (CC50) and 50% effective concentration (EC50) of TS were 59.86 ± 0.3841 µg/ml and 24.29 ± 1.194 µg/ml, respectively. The maximum noncytotoxic concentration (MNTC) of TS on Marc-145 cells was 30 µg/ml. TS could inhibit cell apoptosis induced by PRRSV induced and effectively inhibit PRRSV replication by reducing the expressions of host cellular gene PABP, and significantly inhibiting virus N gene/protein expression. Conclusions: TS possessed anti-PRRSV activity in vitro, and could serve as a potential antiviral drug for PRRSV prevention and control.

Publication: Antiviral Therapy; Type: Original article DOI: 10.3851/IMP2937 Accepted 15 January 2015, published online 22 January 2015 Running title: Tea seed saponins anti-PRRSV in vitro

Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is one of major pathogens to swine. This virus has caused huge economic losses to the swine industry [1,2]. This single stranded RNA virus can cause reproductive failures in pregnant sows, respiratory disorder in piglets, immune suppression and secondary infections [3]. Vaccination is the most commonly used tool for controlling and preventing the syndrome. Modified live-attenuated and inactivated vaccines, however, are insufficient to eradicate infection caused by the PRRSV [4,5]. Hence, novel strategies are needed to control PRRSV infection for the pig industry. Previous studies demonstrated that chemical components extracted from traditional Chinese medicine have anti-cancer, anti-virus, anti-inflammatory effect [6]. Screening of antiviral components from traditional Chinese medicines may lead discovery of new compounds that can be further developed into drugs with activity against PRRSV. PRRSV is a small enveloped, positive-sense single-strand RNA virus with nearly 15kb in length and contains nine open reading frames (ORFs). The conserved nucleocapsid (N) protein, encoded by ORF7, is the most immunogenic protein and plays essential roles in the virus life cycle [7,8]. N protein may also play a role in gene transcriptional regulation in infected cells [9]. Phosphorylation of Coronavirus N protein can facilitate its binding to RNA [10]. Poly (A) binding protein (PABP) is a host cellular protein that enhances transcriptional activity by circularizing mRNAs, and interacts with N protein to facilitate PRRSV replication [11]. Cellular poly (C) binding protein 1 and 2 (PCBP1, PCBP2) were shown to aid PRRSV replication by interacting with Nsp1 [12]. Tea seeds saponins (TS), extracted from tea seeds, possessed antifungal activities [13], antiallergic [14], anti-microbial [15,16] and cardio-protective effects [17]. In addition, they were used for treating burn injuries [18]. Evidence of its antiviral effect is emerging. Hayashi et al. reported that TS could inactivate human type A and B influenza virus, yet the underlying antiviral mechanisms remain unknown [15]. The present study aims to investigate the inhibitory effects of TS on PRRSV and its underlying mechanisms using Marc-145 cells as the model system. We hypotheses that viral N protein and host PABP and cellular poly (C) binding protein play an important role in antiviral action of TS in PRRSV.

Publication: Antiviral Therapy; Type: Original article DOI: 10.3851/IMP2937

Materials and Methods Cell, Virus and TS African green monkey kidney cell line Marc-145 was obtained from China Institute of Veterinary Drug Control. Cells were cultured at 37°C in a 5% CO2 atmosphere in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fatal bovine serum), 100 IU/ml penicillin G and 100 µg/ml streptomycin. PRRSV vaccine (JXAI-R, No. 1012001, Guangdong Dahuanong Animal Health Products Co., Ltd) was propagated in Marc-145 cells. Virus titer was determined by the Reed-Munch formula [19] and 100TCID50/ml was used in this study. Ribavirin was from National Instituted for Food and Drug Control. Tea seeds saponins (TS) was kindly provided by Beijing University of Chinese Medicine. TS was dissolved in 1% DMSO and diluted with DMEM supplemented with 2% FBS.

Cytotoxicity Assay 5

Marc-145 cells were seeded into 96-wells plate at a density of 2×10 /ml about 100 µl per well. At around 90% confluence, medium was removed and cells were subjected to 0, 20, 30, 40, 50, 60, 70, 80 or 90 µg/ml TS in DMEM. Ribavirin was used as control. After 72 h incubation, medium was removed and 20 µl of MTT was added to each well, incubated for 4 h at 37°C. After incubation, MTT solution was discarded and formazan crystals were dissolved in 150 µl DMSO. The absorbance of cell solution in each well was read on an ELISA micro plate reader (ELx808, Gene Co., Ltd., Hong Kong, China) with a 490 nm wavelength and a 630 nm reference wavelength. The percentage (%) of cell survival at various drug concentrations

is

calculated

as:

%

of

cell

survival=(absorbance

of

treated)/(absorbance

of

untreated)×100. The drug concentration leading to 50% of growth inhibition is the cytotoxic concentrations (CC50). This process was implemented using GraphPad Prism

TM

(GraphPad Software, Inc. California,

USA).

Antiviral Assay Antiviral assay was performed with TS (30 µg/ml) and 100 TCID50 PRRSV. 100 µl medium was added to each well in 96-well plate. Controls were set consisting of only cells and cells with virus as well as cells with virus plus ribavirin. When 80-90% cytopathologic effect (CPE) was observed in the group of virus only, MTT assay was carried out to determine the anti-PRRSV effects of TS as previous described [20]. The EC50 was analyzed by GraphPad Prism

TM

(GraphPad Software, Inc. California, USA). The inhibition

ratio was expressed as special value.

The inhibition ratio, (% I) = (

OD test - OD virus )×100% ODcell - OD virus

Publication: Antiviral Therapy; Type: Original article DOI: 10.3851/IMP2937 Time-of-addition Assay Marc-145 cells infected with 100 µl 100TCID50 PRRSV were incubated at 37°C for the different time interval of 1, 2, 4, 6, 8, 10, 12 and 14 h. Cells then were washed with PBS and replaced by culture medium containing TS at the maximum noncytotoxic concentration. The plates were further incubated for 72 h. The antiviral activity of TS was assessed by MTT assay and was measured as the viral inhibitory ratio.

Entry Inhibition Assay In order to examine whether TS inhibit PRRSV entry into cells, virus entry assay was conducted according to previous protocols with minor modification [21]. Briefly, 100% confluent Marc-145 cells grown in 96-well plate was incubated with TS (30 µg/ml) at 37°C for 15 min, 30 min, 1 h, 2 h, 4 h and 6 h, respectively, and then left the plate at 4°C for 1 h. TS was removed from the wells and cell were washed with pre-cold PBS. Subsequently, 100 µl of 100TCID50 PRRSV in the medium was added to each well, incubated at 4°C for 2.5 h, then placed the plate in the incubator at 37°C for 72 h. The inhibition virus entry assay was analyzed by using MTT and the viral inhibitory ratio was determined.

Virucidal Assay In order to assess the direct effect of TS (30 µg/ml) on PRRSV, the TS was mixed with 100TCID 50 PRRSV, and incubated for different times (30, 60, 90, 120 and 150 min) at 37°C, and then the mixture was added onto cells. Virucidal assay was conducted after the cells were incubated for 72 h.

Apoptosis Assay Apoptosis was analyzed with AnnexinV- EGFP Kit (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, P.R. China) as previously described with some modifications [22]. Briefly, EGFP-conjugated Annexin V (AV) and propidium iodide (PI) were added to the cells, the cell cultures were then incubated 15 min at room +

-

temperature in the dark. Only green fluorescence around cell membrane (AV PI ) was considered as early apoptotic cells. Late apoptotic cells were shown by green fluorescence around cell membrane and red +

+

fluorescence in nucleus (AV PI ).

Absolute Quantification RT-PCR 100TCID50 PRRSV was incubated with Marc-145 cells for 3 h in a 6-well plate, then the virus was discarded, the cells were harvested at 4, 14, 20, 36 and 52 h after incubating with TS (30 µg/ml, 2 ml). Total cellular RNA was extracted from Marc-145 cells by using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. cDNA was generated with PrimeScript

®

RT Master Mix

(TaKaRa) and Real-time PCR was used to amplify the N gene using a Mx3000PTM QPCR System (Stratagene, USA). The cycling conditions were as follows: 95°C 10 min, followed by 40 cycles of 95°C for 5 s, 61°C for 30 s and 72°C for 30 s. A standard curve was generated using Puc19-N recombinant

Publication: Antiviral Therapy; Type: Original article DOI: 10.3851/IMP2937 plasmid. To confirm the specific amplification, melt-curve analysis of the real-time PCR products was performed. The PRRSV RNA copies were determined by linear extrapolation against the standard curve.

Relative Quantification RT-PCR Cells were treated the same as those in the section of Absolute quantification RT-PCR, except cells were collected at 24 h and 48 h post-treatment. Five sets of primers were designed for this RT-PCR (Table. 1). The PCR condition were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s per cycle, 56°C -

Ct

for 30 s and 72°C for 20 s. The expression levels of cell genes were calculated using 2  methods.

Western Blot Analysis Cells were incubated with 100TCID50 PRRSV in 6-well plate. After 3-h-icubation, the virus was discarded, the concentrations of TS (30 µg/ml, 20 µg/ml or 10 µg/ml) were added onto plate. Cells were harvested at 24, 48 and 72 h later. Total protein was extracted using a total protein extraction Kit (Applygen, P. R. China) following the manufacturer's instruction. Samples of 20 g protein were subjected to 12% SDSPAGE and transferred to PVDF (0.22 µm, Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in TBST for 2 h at room temperature the membrane was incubated with anti-PRRSV N protein monoclonal antibody (1:1000) and anti--actin antibody at 4°C overnight. Protein bands were exposed using ECL and presented to autoradiography film. -actin was used as the loading control.

Statistical Analysis Data were expressed as mean ± SD. The statistical significance was determined by Student’s t-test or one-way ANOVA. Results were considered to be significant if p less than 0.05. *: P