Studies On Immuno-regulation And Antivirial Activity Of Three Botanic Polysaccharides Against PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS) firstly recognized in North America in 1987. PRRS is characterized by reproductive failures in late-term pregnant sows, high mortality in unweaned piglets and respiratory problems in pigs of all ages. PRRS has been a major health problem to swine herds worldwide and causes great economic loss in pork production industry since it first appeared. Many strategies aimed to control and eradicate PRRS have not always been effective, possibly due to the characteristic properties of the virus such as genetic and antigenic variations, subclinical and persistent infections, viremia with concurrent antibody, and antibody-dependant enhancement. Therefore, development of safer and more effective immunization remains a big challenge.Polysaccharides possess immunity enhancing and antiviral activities. In this study, three herbal polysaccharides were selected as immunoregulationer, their antiviral and immune-regulating abilities were investigated through a series of experiments in vitro and vivo. The purpose is to offer the theory basis for developing polysaccharides as immunopotentiators.1. Three herbal polysaccharides were extracted respectively from Astragalus, isatis root and Chinese yam through water-extraction and ethanol-precipitation. The total contents (%) of Astragalus polysaccharides (APS), isatis root polysaccharides (IRPS), and Chinese yam polysaccharides (CYPS) were 65.8, 56.7 and 73.5 respectively, measured by Vitriol-anthracene keton.. The total extractive rates (g·kg^-1) of them were 37.4, 59.1 and 53.6 respectively. To evaluate the antiviral activity and mechanism of the three polysaccharides, APS, IRPS and CYPS were respectively applied to Marc-145 cell culture before, after and simultaneous with PRRSV respectively. The cytopathic effect (CPE) were observed and the OD values of the cultures were determined by neutral red colorant absorption method to evaluate the effects of three polysaccharides against virus infection. The results showed that three Chinese herbal polysaccharides could decrease CPE lesions and inhibit virus from infecting the cells in a dosage dependent mode when the polysaccharides were added early to PRRSV, APS and IRPS also displayed antiviral activity when simultaneously added with PRRSV, but the three polysaccharides had no effect on the virus in the infectural cells.2. Effects of the three polysaccharides on immunity of mice were studied by testing the intraperitoneal macrophages function, the formation of haemolytic plaque and seral hemolysin. The results showed that the three herbal polysaccharides could improve the function of intraperitoneal macrophages, raise the level of haemolytic empty spots and seral hemolysin, and the optimal effective dosages were respectively 30mg·kg^-1 of APS , 20mg·kg^-1 of IRPS and 50mg·kg^-1 of CYPS.3. In order to study the effects of the three polysaccharides on specific-antibody levels in response to PRRS inactivated or live virus vaccine, piglets were inoculated with vaccines along with APS, IRPS and CYPS at high or low dose respectively. The results showed that: one polysaccharides extract had different effects on immuno-regulation when applied with different types of vaccine, and the potential were closely correlated to its dose. IRPS at high dosage could enhance the antibody titers induced by inactivated and live PRRS virus vaccine, whereas it just enhanced the antibody titer induced by inactivated vaccine but decreased that by live virus vaccine at low dosage .APS could enhance the antibody titer for inactivated vaccine, the high dose being more potent, but it showed opposite effects on live virus vaccine, the higher dose, the lower titer. CYPS could enhance the antibody titer for inactivated vaccine prominently, the low dose being better, but decreased the titer for live virus vaccine.4. Effects of the three polysaccharides in vitro on proliferation of swine splenic lymphocytes and secretion of cytokines and NO were studied. The results showed that at suitable dosages the three herbal polysaccharide could stimulate proliferation of splenic lymphocyte induced by ConA or LPS significantly (p<0.01) in vitro, and effects of three polysaccharides were related to their dosage. APS and CYPS could stimulate excretion of NO at any dose, and CYPS effected more strongly. IRPS could improve excretion of NO at low dose but inhibit at high dose. The three polysaccharide could promote the production of IL-4 (p<0.01) , and APS was the best. Remarkable upregulation of IFN-γco-induced by ConA and polysaccharide were observed in all three polysaccharides, and IRPS was the best , CYPS being the next. The boosting effection of polysaccharides on IFN-γand IL-4 was related to their dose. In contrast, the three polysaccharides appeared to inhibit the secretion of IL-2 significantly (p<0.05)5. Effects of the three polysaccharides on T subpopulations in the immune response to PRRS inactivated or live virus vaccine were studied. The results showed that: in the inactivated vaccine co-inoculating groups, the three polysaccharides could improve the proportion of CD3⁺ and CD8⁺ in pheripheral blood of piglets, CYPS and IRPS being better than APS. In the live vaccine co-inoculating groups, the three polysaccharides improved the proportion of CD8⁺ at the early stage, IRPS and APS being better than CYPS, but all had no influence on CD3⁺ and CD4⁺. It indicated that the effect of the herbal polysaccharides on T subpopulations in the immune response to PRRS were related to the type of vaccine. The results would reveal the immuno-regulation of polysaccharides at cellular level in vivo.6. Effects of the three polysaccharides on IFN-γmRNA expression in the immune response to PRRS live virus vaccine were studied by real-time fluoresence quantitative PCR (RT-FQ-PCR). The results showed that APS and CYPS could down-regulate the IFN-γmRNA expression in porcine PBMC, especially at the early stage, while IRPS affected the IFN-γmRNA expression dose-dependently. High dose IRPS promoted IFN-γmRNA expression and low dose depressed it. It would reveal the immuno-regulation of polysaccharides at nucleic acid level in vivo.