Inhibiting mechanism of Alpiniae oxyphyllae fructus polysaccharide 3 against the replication of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) causes diarrhea, vomiting, and death in piglets. Our previous study has revealed the anti-PEDV activity of Alpiniae oxyphyllae fructus polysaccharide 3 (AOFP3). However, it is still unknown whether AOFP3 can inhibit the replication of PEDV. Therefore, the effect of AOFP3 on PEDV replication was investigated in the present study, along with analysis of viral RdRp activity and expression of hnRNP A1 by RNA polymerase activity assay in vitro, RIP assay, and Western blotting. The results showed that both the PEDV gene and protein levels in IPEC-J2 cells decreased with AOFP3 treatment. In addition, AOFP3 significantly reduced PEDV's replication by down-regulating the activity of PEDV RdRp and reducing the expression of hnRNP A1, whereas only the bind of RdRp to PEDV 3'UTR was inhibited in AOFP3 treated cells.

Detection of RNA-dependent RNA polymerase of porcine epidemic diarrhea virus.

The RNA synthesis of porcine epidemic diarrhea virus (PEDV) is a sophisticated process performed by a multilingual viral replication complex, together with cellular factors. A key enzyme of this replication complex is RNA-dependent RNA polymerase (RdRp). However, there is limited knowledge about PEDV RdRp. In our present study, a polyclonal antibody against RdRp was prepared by using a prokaryotic expression vector pET-28a-RdRp to study the function of PEDV RdRp and provide a tool to investigate PEDV pathogenesis. In addition, the enzyme activity and half-life of PEDV RdRp were investigated. The result showed that the polyclonal antibody against PEDV RdRp was successfully prepared and was able to be used to detect PEDV RdRp by immunofluorescence and western blotting. Additionally, enzyme activity of PEDV RdRp reached nearly 2 pmol/µg/h and the half-life of PEDV RdRp was 5.47 h.

Inhibition of porcine epidemic diarrhea virus by Alpiniae oxyphyllae fructus polysaccharide 3.

Porcine epidemic diarrhea virus (PEDV) is a deadly pathogen that still plagues suckling piglets. However, there is still no anti-PEDV drug available in clinics. To develop potential anti-PEDV drugs, the antiviral activity of Alpiniae oxyphyllae fructus polysaccharide 3 (AOFP3) against PEDV infection in IPEC-J2 cells were assessed in our present study. The structural characterization of AOFP3 was studied by using HPAEC, GC-MS, FT-IR and NMR techniques. At the same time, the anti-PEDV activity of AOFP3 was investigated by performing RT-qPCR, Western blot and immunofluorescence assays. The results showed that AOFP3 (44.4 kDa) was composed of glucose and galacturonic acid at a molar ratio of 77.54:22.46 and consisted of →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, T-α-D-Glcp-(1→ and →4)-α-D-GalAp-(1→. AOFP3 significantly decreased PEDV titer in IPEC-J2 cells and prevented cellular damage of IPEC-J2 cells caused by PEDV infection. Furthermore, AOFP3 showed an antioxidative activity in inhibiting PEDV reproduction. Therefore, AOFP3 was expected to be a material of anti-PEDV drug.

Characterizations of glucose-rich polysaccharides from Amomum longiligulare T.L. Wu fruits and their effects on immunogenicities of infectious bursal disease virus VP2 protein.

The aim of this study is to explore the characterization of Amomum longiligulare T.L. Wu fruits polysaccharide (ALP) and their immune enhancement effects. Two homogeneous polysaccharides (ALP1 and ALP2) were isolated from the fruits. The structural characterization results showed that ALP1 (26.10 kDa) and ALP2 (64.10 kDa) were both mainly composed of glucose. Furthermore, ALP1 was consisted of (1,2)-α-D-Glcp, (1,2,3)-α-D-Glcp and T-α-D-Glcp, while ALP2 was consisted of T-α-D-Glcp, (1,3)-α-D-Glcp and (1,3,6)-α-D-Glcp. Afterwards, the immune enhancement effects of two polysaccharides were evaluated by determining their effects on immunogenicities of infectious bursal disease virus (IBDV) VP2 protein. Chickens were immunized with IBDV VP2 protein accompanied with ALP1/ALP2. And the results indicated both ALP1 and ALP2 promoted the weights and bursa of fabricius indexes of chickens. In addition, both two polysaccharides increased specific IBDV antibody levels, while ALP1 possessed higher immune enhancement ability and was expected to be an adjuvant for IBDV VP2 protein.

Structural characterization of Alpiniae oxyphyllae fructus polysaccharide 2 and its activation effects on RAW264.7 macrophages.

Polysaccharides are important components of Alpiniae oxyphyllae fructus that have been shown to exhibit significant immunomodulatory activity in our previous study. However, whether and how A. oxyphyllae fructus polysaccharides (AOFP) affect macrophages has not been determined. To further study the immunomodulatory activity of AOFP, the effect of AOFP on RAW264.7 cell activation was investigated in the present work. The results showed that AOFP2 significantly increased the phagocytic activity of RAW264.7 macrophages. AOFP2 promoted the secretion of TNF-α, IL-6, IL-10, TGF-ß, NO and iNOS and enhanced the Th2-type immune response via its activation effect on macrophages. Additionally, the structure of AOFP2 was characterized in the present study, as the structural features of polysaccharides determine their biological activities. AOFP2 was only composed of glucose, exhibiting an average molecular weight of 44.3 kDa. Furthermore, the infrared spectroscopy, methylation and nuclear magnetic resonance results indicated that AOFP2 consisted of â†’ 4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1 â†’ and T-α-Glcp.

Okra polysaccharide-2 plays a vital role on the activation of RAW264.7 cells by TLR2/4-mediated signal transduction pathways.

Polysaccharide is the main active component of okra (Abelmoschus esculentus L.) and it can effectively stimulate the activation of macrophages. However, the immune regulatory mechanism is still not clear. Therefore, the present study aimed to reveal the possible mechanism by investigating the effect of okra polysaccharide-2 (RPS-2) on Toll-like receptor (TLR) 2/4-mediated signal transduction pathways in RAW264.7 murine macrophage cells. In order to confirm whether RPS-2 stimulated macrophages activation via TLR2 or TLR4, RAW264.7 murine macrophage cells were pretreated with TLR2/4 inhibitors for 1 h before RPS-2 treatment, and then the NO, IL-10, TNF-α levels were tested. The results indicated that both TLR2 and TLR4 were the keys of immune regulatory effect of RPS-2. Afterwards, the effect of RPS-2 on NF-κB and MAPKs signaling pathways were studied by western blot analysis. It showed RPS-2 induced the phosphorylation of p65, IκBα, p38, ERK1/2 and JNK. At the same time, the specific inhibitors reduced these phosphorylation levels as well as NO, IL-10 and TNF-α amounts. In a word, RPS-2 activated macrophages by NF-κB and MAPKs signal transduction pathways.

Antiviral activity against porcine epidemic diarrhea virus of Pogostemon cablin polysaccharide.

ETHNOPHARMACOLOGICAL RELEVANCE: The dry overground parts of Pogostemon cablin (Blanco) Benth. is widely used in China as a traditional Chinese medicine for the treatment of diarrhea, vomiting, nausea and fever. Polysaccharide is an important component of Pogostemon cablin (Blanco) Benth. but has not been studied. Pogostemon cablin (Blanco) Benth. is used to treat porcine epidemic diarrhea. But it is not known whether Pogostemon cablin polysaccharides (PCPs) has the antiviral activities against porcine epidemic diarrhea virus (PEDV). AIM OF THE STUDY: The purpose of present study is to investigate the structural characterization and the anti-PEDV activities of PCPs. MATERIALS AND METHODS: PCPs were prepared by water extraction and alcohol precipitation method and purified with DEAE-52 cellulose column and Sephadex G-100 column. Then, the structural characterization of the polysaccharides including the infrared spectrum, molecular weight and monosaccharide composition were analyzed. Afterwards, the antiviral effect of PCPs against PEDV on IPEC-J2 cells was studied by MTT method and real-time PCR method. Additionally, the effects of PCPs on PEDV adsorption, penetration and replication were analyzed by real-time PCR method. Furthermore, we also investigate whether the anti-oxidative effects of PCPs were important to the anti-PEDV activities. RESULTS: Four polysaccharides were obtained and named as PCP1.1 (31.3 kDa), PCP1.2 (3.5 kDa), PCP2.1 (9.1 kDa) and PCP2.2 (8.3 kDa). PCP1.1, PCP1.2 and PCP2.1 were composed of fucose, arabinose, galactose, glucose, mannose, galacturonic acid and glucuronic acid; and PCP2.2 was composed of arabinose, galactose, glucose, galacturonic acid and glucuronic acid. All PCPs showed anti-PEDV activities. PCP1.1 and PCP1.2 inhibited PEDV replication, while PCP2.1 and PCP2.2 inhibited PEDV penetration and replication. All PCPs showed anti-oxidative effects, which were important to the anti-PEDV activities. CONCLUSIONS: The treatment effect of Pogostemon cablin (Blanco) Benth. on porcine epidemic diarrhea might be related to the anti-PEDV effect of PCPs. Furthermore, the anti-oxidative effects of PCPs play important roles in their antiviral activities against PEDV.