| Porcine reproductive and respiratory syndrome(PRRS) is one of the most economically devastating infectious diseases in the worldwide pork industry caused by porcine reproductive and respiratory syndrome virus(PRRSV) and characterized with respiratory distress, high mortality in piglets and reproductive failure in sows. PRRSV was firstly discovered in United States in 1987 while it was firstly reported in 1996 in China. The highly pathogenic PRRS(HP-PRRS) emerged in China in 2006 and it quickly spread to some of Southeast Asian countries. The current strategy to prevent and control PRRS is vaccination. However, vaccination can not fully provide the guarantee of safety and effects because of high variabilities of prevalent PRRSV strains. Furthermore, till today, there is no approved drugs for clinically dealing with PRRS. Therefore, it's an urgent requirement to develop novel anti-PRRSV drugs. Traditional Chinese medicines have been played a very important role in the defense of viral diseases. It is an efficient strategy to develop the anti-PRRSV agents from the natural compounds.Procyanidins(PC) and epigallocatechin gallate(EGCG), belonged to the family of tea polyphenols compounds, are well-known as antioxidants. PC and EGCG, which has been used wildly in food, cosmetic and health care industries, possesses many biologic activities such as antioxidation, antisenility, antivirus, antibiosis and anticancer. In this thesis, the antiviral activities of PC and EGCG against PRRSV in vitro were evaluated and the underlying mechanism of action was studied.In this study, the experimental model of Marc-145 cells infected with PRRSV was established firstly. Out of eleven tea polyphenols, PC and EGCG showed significant antiviral activities against PRRSV infections through activity screening of Indirect Immunofluorescence Assay(IFA). Based on this finding, a systemic investigation was implemented on anti-PRRSV activities of PC and EGCG. m RNA levels of non-structural protein 9 and N protein levels of PRRSV were measured by real-time PCR and Western blot, respectively. The antiviral effects of PC and EGCG at 48 h post infection(pi) and at different time points within 48 h pi were evaluated. Furthermore, time course experiments by adding PC or EGCG at different time points pi were carried out to investigate which stages of the life cycle of PRRSV was intervened. The effects of pretreatments of PC or EGCG with PRRSV or Marc-145 cells were also determined. The purpose of all these experiments was to disclose the preliminary underlying antiviral mechanisms of PC and EGCG against PRRSV.The results showed that PC and EGCG could significantly inhibit the proliferation of PRRSV at 48 h pi at the range of 5~40 ?g/m L in a dose-dependent manner. Especially, the inhibitory effect of 40 ?g/m L PC was much better than that of 40 ?g/m L ribavirin. Forty ?g/m L of PC and EGCG could also suppress the synthesis of the m RNA of NSP9 and N protein at 6, 12, 24, 36 and 48 h pi, respectively. Results indicated that PC and EGCG blocks the stages of attachment, internalization and release of PRRSV in a dose-dependent manner. Moreover, PC and EGCG at 40 ?g/m L inhibited the replication phase of PRRSV. When PRRSV was pretreated with PC or EGCG at the range of 5~40 ?g/m L, the virulence of PRRSV was significantly impaired. PRRSV could normally infect Marc-145 cells which was pretreated by PC or EGCG, indicating that the two compounds have no activity of sealing up the cellular receptors for the virus..To the best of our knowledge, this study is the first study to discover the antiviral activities of PC against PRRSV and find that the antiviral mechanisms of PC and EGCG are related to their inhibitory effects on stages of attachment, internalization, replication and release of PRRSV and the ability of directly damaging the virus particles.The present study may provide valuable insights and experimental basises for the development of novel antiviral drugs against PRRS. |
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