Yaogui Sun

Anti-PRRSV effect and mechanism of sodium tanshinone IIA sulfonate in vitro

This experiment was conducted to study the antiviral activities of sodium tanshinone IIA sulfonate (STS) against porcine reproductive and respiratory syndrome virus (PRRSV) and its mechanism. Anti-PRRSV activities of STS were observed on Marc-145 cells by using visualization of cytopathologic effect assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, and its antiviral mechanism was determined by time-of-addition assay, adsorption inhibition assay, and virucidal assay. The results showed that STS could reduce the damage of PRRSV to Marc-145 cells, with the inhibition ratio exceeding to 100%, at the maximum non-cytotoxic concentration. The time-of-addition and virucidal assays indicated that the anti-PRRSV activities of STS could be due to inhibiting the virus replication or/and inactivating the virus directly. The inhibition of the virus attachment was not discovered in adsorption inhibition assay. The results proved that STS had strong anti-PRRSV activity and encouraged for further exploration of STS.

Matrine displayed antiviral activity in porcine alveolar macrophages co-infected by porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

The co-infection of porcine reproductive respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) is quite common in clinical settings and no effective treatment to the co-infection is available. In this study, we established the porcine alveolar macrophages (PAM) cells model co-infected with PRRSV/PCV2 with modification in vitro, and investigated the antiviral activity of Matrine on this cell model and further evaluated the effect of Matrine on virus-induced TLR3,4/NF-κB/TNF-α pathway. The results demonstrated PAM cells inoculated with PRRSV followed by PCV2 2 h later enhanced PRRSV and PCV2 replications. Matrine treatment suppressed both PRRSV and PCV2 infection at 12 h post infection. Furthermore, PRRSV/PCV2 co- infection induced IκBα degradation and phosphorylation as well as the translocation of NF-κB from the cytoplasm to the nucleus indicating that PRRSV/PCV2 co-infection induced NF-κB activation. Matrine treatment significantly down-regulated the expression of TLR3, TLR4 and TNF-α although it, to some extent, suppressed p-IκBα expression, suggesting that TLR3,4/NF-κB/TNF-α pathway play an important role of Matrine in combating PRRSV/PCV2 co-infection. It is concluded that Matrine possesses activity against PRRSV/PCV2 co-infection in vitro and suppression of the TLR3,4/NF-κB/TNF-α pathway as an important underlying molecular mechanism. These findings warrant Matrine to be further explored for its antiviral activity in clinical settings.

In vitro evaluation of antiviral activity of tea seed saponins against porcine reproductive and respiratory syndrome virus.

BACKGROUND Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major swine pathogens. This virus causes immune suppression and other secondary infections, leading to significant economic losses in the swine industry. Tea seed saponins (TS) are a natural extract from tea seeds with anti-cancer, anti-inflammatory 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. 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 analysed 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 non-cytotoxic concentration of TS on Marc-145 cells was 30 μg/ml. TS inhibited PRRSV-induced cell apoptosis and effectively inhibited PRRSV replication by reducing the expression of host cellular gene PABP, and significantly inhibited 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.

Antiviral effects of the constituents derived from Chinese herb medicines on infectious bursal disease virus

Abstract Context: The prevalence of infectious bursal disease has brought about enormous financial losses to the world poultry industry. Chinese herb medicines can provide valuable materials for discovery and development of new drugs. Objective: To screen constituents derived from Chinese herb medicines for their antiviral activity against infectious bursal disease virus (IBDV) in vitro. Materials and methods: Twenty constituents derived from Chinese herb medicines and B87 strain of IBDV were used. The 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) were determined by visualization of cytopathologic effect (CPE) and 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) test on chicken embryo fibroblast. Selectivity index (SI) and inhibition ratio (%I) were calculated from the data obtained from the MTT test. Results: Antiviral assays showed dipotassium glycyrrhizinate and ligustrazine hydrochloride among the 20 constituents tested exhibited significant inhibitory activity against IBDV in a dose-dependent manner. EC50 of dipotassium glycyrrhizinate and ligustrazine hydrochloride were 663.2 ± 268.4 and 92.52 ± 21.13 µg/mL, and SI were >4.52 and >21.62, respectively. The time-of-addition and virucidal assay indicated that anti-IBDV activity of the two constituents could be due to their inhibiting virus replication and/or inactivating virus directly. The inhibition of virus attachment was not observed in the adsorption inhibition assay. Dipotassium glycyrrhizinate and ligustrazine hydrochloride exhibited more than 70% and 80% inhibition of IBDV, respectively, at the maximum safe concentration. Discussion and conclusion: We believe that dipotassium glycyrrhizinate and ligustrazine hydrochloride can be used to develop a new anti-IBDV compound, and it is worth applying the constituents in clinical practice.

The cytological observation of immune adherence of porcine erythrocyte.

Abstract The immune adherence (IA) between the porcine erythrocytes and the opsonized Escherichia coli carried green fluorescent protein gene (GFP-E.coli) were detected by the fluorescence microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) with an attempt to verify the existence of IA between the porcine erythrocytes and complemented-opsonized microbes. Under fluorescence microscopy, GFP-E.coli opsonized by fresh rabbit serum complement adhered to the erythrocytes and could not be detached by PBS washing, and no IA was observed between the erythrocytes and nonopsonized GFP-E.coli after co-incubation. SEM and TEM also revealed the existence of IA between the serum complement-opsonized GFP-E.coli membrane and the erythrocyte membrane. The partial complement receptor type 1 (CR1)-like gene from porcine was generated by RT-PCR and rapid amplification of cDNA 3’ end (3’ RACE) (157bp and 578bp), both of which have high similarity with published mammals CR1 gene. The sequences were spliced based on homology comparison and submitted to GenBank (GenBank Accession No. JX033989). These results indicated that the porcine erythrocytes were able to bind to the opsonized microorganisms. Furthermore, the sequencing results confirmed that the CR1-like gene exists in porcine.

Antiviral activities of natural compounds derived from traditional chinese medicines against porcine circovirus type 2 (PCV2)

Twenty natural compounds isolated from traditional Chinese medicines were examined for their antiviral activities against PCV2 in vitro. Antiviral activity was analyzed by the assays of blocking of virus entry, inhibition of virus replication and inactivation of virus particles in the virus suspension. 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) for each compound were determined by using the MTT method and immunofluorescence assay (IFA), respectively. The results obtained from the inhibition of virus replication and inactivation of virus particles showed that the maximum inhibition rates for matrine and scutellarin were 57 and 72.69% and their SI were 29.87 and 18.66, respectively. However, the results from the assays of inhibition of virus entry and inhibition of virus replication demonstrated that both aesculetin and arecoline hydrochloride stimulated PCV2 replication, with the maximum stimulation rate of 387.3 and 219.4%. In summary, both matrine and scutellarin had antiviral activity against PCV2, while aesculetin and arecoline hydrochloride promoted PCV2 infection replication in vitro.

Screening compounds of Chinese medicinal herbs anti-Marek's disease virus.

Abstract Context: Marek’s disease (MD) seriously threatens the world poultry industry and has resulted in great economic losses. Chinese medicinal herbs are a rich source for lead compounds and drug candidates for antiviral treatments. Objective: To investigate the anti-MDV activity and mechanism of 20 compounds extracted from Chinese medicinal herbs. Materials and methods: Antiviral assay, time of addition experiments, and virucidal assay were performed on chicken embryo fibroblast cells. The 50% cytotoxic concentration and 50% effective concentration were determined and, accordingly, selectivity index and inhibition ratio were calculated. Results: Antiviral assay showed dipotassium glycyrrhizinate (DG) and sodium tanshinone IIA sulfonate (STS) exhibited significantly inhibitory activity against MDV in a dose-dependent manner. EC50 of DG and STS were 893.5 ± 36.99 µg/mL and 54.82 ± 2.99 µg/mL, and selective index (SI) were >3.36 and >9.12, respectively. Time of addition experiment and virucidal assay demonstrated DG inhibited viral replication in the full replication cycle and inactivated MDV particles in non-time-dependent manner, but STS interfered with the early stage of MDV replication and inactivated MDV particles in a time-dependent manner. Moreover, both DG and STS promoted apoptosis of cells infected by MDV. Discussion and conclusion: DG and STS have great potential for developing new anti-MDV drugs for clinic application.

Expression and Purification of Soluble Porcine Cystatin 11 in Pichia pastoris

Cystatin 11 (CST11) belongs to the cystatin type 2 family of cysteine protease inhibitors and exhibits antimicrobial activity in vitro. In this study, we describe the expression and purification of recombinant porcine CST11 in the Pichia pastoris system. We then assess its antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis by liquid growth inhibition assay. Kinetic studies indicate that the recombinant porcine CST11 has high potency against E. coli and S. aureus. Scanning electronic microscope analysis showed that CST11 might be targeting the bacterial membrane and, thus, could potentially be developed as a therapeutic agent for inhibiting microbe infection without the risk of antibiotic resistance.

Cloning and bioinformatics analysis of a full-length cDNA of porcine CR1-like gene

Jing Cheng1, Junbing Jiang1, Junxing Zhao1, Zhirui Wang2,3, Yaogui Sun1, Haili Ma1, Kuohai Fan1, Wei Yin1, Na Sun1, Zhiwei Wang1, Xin Zhao1, and Hongquan Li1* College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02148, USA MGH-DF/HCC Recombinant Protein Expression and Purification Core, Boston, MA 02148, USA *Correspondence address. Tel/Fax: þ86-354-6288409; E-mail: livets@163.com

Expression and purification of the recombinant porcine NK-lysin in Pichia pastoris and observation of anticancer activity in vitro

ABSTRACT Natural killer (NK)-lysin, a broad-spectrum antimicrobial peptide, has antitumor and antibactericidal activities against both gram-positive and gram-negative bacteria. In this study the recombinant porcine NK-lysin was expressed and purified in the Pichia pastoris system, and then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to assess its anticancer activity in vitro. The results showed that the recombinant porcine NK-lysin possesses potent antitumor activity against the human hepatocellular carcinoma cell line SMMC-7721 in a time- and dose-dependent manner, but has negligible hemolysis activity against human erythrocytes. Scanning electronic microscopy was used to directly observe the ultrastructure of SMMC-7721 cells treated with NK-lysin; untreated cells showed lamellipodia and filopodia scattered with the cell surface, with good cell–cell contacts among neighboring cells. In contrast, treated tumor cells exhibited marked alterations in cell morphology, and cell–cell contacts disappeared among neighboring cells. Compared with the untreated tumor cells, the tumor cells treated with NK-lysin for 12 and 24 hr were suppressed for the expression of fascin 1. Thus, the recombinant porcine NK-lysin potentially could be developed as a therapeutic agent for inhibiting tumor growth.