Abstract：Swine transmissble gastroenteritis(TGE) is a kind of hyperinfected viral disease in pigs (Sus scrofa) induced by Swine transmissble gastroenteritis virus (TGEV), and leads to death of large numbers of the pigs. The present study aimed to evaluate the effect of Polygonum cillinerve polysaccharide (PCP) on inhibition of TGEV. Based on the detection results of TCID50 of TGEV and maximum cytotoxic concentration (TC0) of PCP on in vitro growth of swine testicular (ST) cells, the PCP was pro-seeded, co-seeded or post-seeded with TGEV in ST cells, then the anti-virus adsorption, inactivation and inhibition of the proliferation of PCP were analyzed by 3- (4,5- Dimethylthiazol-2- yl)-2,5- diphenyltetrazolium bromide (MTT) method and quantitative Real-time PCR (qRT-PCR). The detection of in vitro viral infection indicated that the value of TCID50 on TGEV was 10-6.25. After 72 h in vitro culture, ST cells were well-grown without the cytotoxic response when treated them with PCP at concentration of lower than 20 μg/mL compared with the blank controls, which determined the TC0 of PCP was 20 μg/mL. According to the results of MTT, under the maximum safe concentration (20 μg/mL) of PCP, the anti-adsorption effect of PCP on TGEV in vitro was the best, the inhibition rate was 85.2%, the inhibition rate on in vitro virus proliferation was 78.4%, while the inhibition rate of direct inactivation to TGEV was 74.4%, and the inhibition rate was positively correlated with the concentration. Moreover, qRT-PCR detection indicated that 3 administration methods of PCP could significantly reduce relative TGEV N mRNA expression at the transcriptional level (P＜0.01) compared with the virus control, and with a dose-dependent characteristic. The results suggested that all 3 administration methods of PCP could inhibit the proliferation of TGEV at ST cells in vitro, and the prevention effect of PCP was the best. This work demonstrated the in vitro anti-virus effect of PCP on TGE, and can provide some basis on resource exploitation and multiple utilization of Polygonum cillinerve.
[1]Compile Group of Complication of Countrywide Herbal Medicine of China..Complication of Countrywide Herbal Medicine of China. Beijing: People’s Medical Publishing House.383(全国中草药汇编编写组. 1996.全国中草药汇编, 上册, 北京:人民卫生出版社, 383)[J]., 1996, :-[2]Cui J J, Yuan J F and Zhang Zh Q,.Extraction and Biological Activity of Crude Polysaccharide from Polygonum ciliinerve. Natural Product Research and Development, 19:960-964 (崔军见, 原江锋, 张志琪, 2007.朱砂七粗多糖的提取及生物活性的研究.天然产物研究与开发, 19:960-964)[J]., 2007, (19):960-964[3]Chu X L, Su J Q and Wei X B, 2009.Latest research progress of antiviral herbs. Journal of Chinese Veterinarian medicine, 28(2)26-28(褚秀玲, 苏建青, 韦旭斌, 2009.中药抗病毒最新研究进展.中兽医医药杂志, 28(2)26-28)[J]., 2009, 28(2):26-28[4]Cui J J, Yuan J F and Zhang Zh Q, 2010.Anti-oxidation activity of the crude polysaccharides isolated from Polygonum cillinerve (Nakai) Ohwi in immune suppressed mice 11; 132(2):512-7.[5]Dong L J, Zhang Y M, He L, Cheng M, Liu W and Lin Zh, 2012.Establishment of RT-PCR for Transmissible Gastroenteritis Virus. Journal of Northwest A＆F University Nat.Sci.Ed, 40(20)13-19(董玲娟，张彦明，何雷，程敏，刘伟，林鸷，2012.猪传染性胃肠炎病毒荧光定量RT-PCR检测方法的建立.西北农林科技大学学报(自然科学版), 40(20)13-19)[6]Ding L, Huang Y, Qian D, Dong F, Zhao X M, Zhang W L, Xu X G and Tong D W, 2014.TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling. Biochemical and biophysical research communications 445(2): 497-503.[7]Enjuanes L, Almazan F and Ortego J, 2003.Virus-based vectors for gene expression in mammalian cells: Coronavirus. In: Makrides SC, ed. Gene Transfer and Expression in Mammalian cells. Elsevier ScienceB.V:151–168[8]Faccin-Galhardi L C, Yamamoto K A, Ray S, Ray B, Carvalho Linhares R E and Nozawa C.J, 2012.The in vitro antiviral property of Azadirachta indica polysaccharides for poliovirus. Ethnopharmacol, 142(1): 86-90.[9]Meng F and Ren X.2011. Characterization and utility of monoclonal antibodies against spike protein of transmissible gastroenteritis virus. Letters in Applied Microbiology, (52):201-207[10]Guo H L, Zhao Q, Zhao Q, Tian X P, Zhao Q, Hu Y, Zhang S H and Zhang X Sh, 2006.The experimental research of Polygonum cillinerve (Nakai) Ohwi inhibition of influenza virus A, Shaanxi traditional Chinese medicine, 27（3）351-353(郭惠玲，赵琦，田小平，赵勤，胡悦.，张思户，张晓双.2006.朱砂七抑制流感病毒A的试验研究.陕西中医，27（3）351-353)[11]Gong G Q, Gong G H, Qian Zh Y, Zhou Sh, 2005.Inhibitory Activity of Luteolin against Transmissible Gastroenteritis Virus. Journal of China Pharmaeutical University., 36( 5) : 453-456(龚国清, 龚国华, 钱之玉, 周曙, 2005.木犀草素对猪传染性胃肠炎病毒的抑制作用.中国药科大学学报, 36( 5) : 453-456)[12]Gao Ch Ch, Deng L L, Yin X, Gao Y and Wei P, Effect of probiotics from porcine gastrointestinal tract on inhibition of transmissible gastroenteritis coronavirus.Chinese Journal of Preventive Veterinary Medicine, 35(9)715-719(高长春, 邓玲玲, 尹鑫, 高雅，魏萍, 2013.猪源益生菌体外抗猪传染性胃肠炎病毒作用的研究.中国预防兽医学报, 35(9)715-719)[13]Gu J P, Yue X W, Xing R, Li C Y and Yang Z B.2012. Progress in genetically engineered vaccines for porcine transmissible gastroenteritis virus. Revue Méd. Vét, 3(163):107-1[14]Huang X Y, Wang D Y, Hu Y L, Lu Y, Guo Zh H, Kong X F and Sun J L, 2008.Effect of sulfated astragalus polysaccharide on cellular infectivity of infectious bursal disease virus. International Journal of Biological Macromolecules, 42:166-171.[15]H S Mason and M M Herbst-Kralovetz, 2012.Plant-Derived Antigens as Mucosal Vaccines. Mucosal Vaccines, (354):101-120[16]He L, Zhang Y M, Dong L J, Cheng M, Wang J, Tang Q H and Wang G, 2012.In vitro inhibition of transmissible gastroenteritis corona virus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene. Virology Journal, 9(1): 1-9.[17]Ji L N, Zhang W M, Qing S Zh, Bai J L and Shi X M, 2013, The extraction and separation of Polygonum Cillinerve polysaccharide and antibacterial experiment in vitro.Heilongjiang animal husbandry and veterinary science and technology, 04, 117-119(姬丽娜，张为民，卿素珠，白建龙，史秀明. 2013, 朱砂七多糖的提取分离及体外抑菌试验.《黑龙江畜牧兽医》科技版, 04, 117-119)[18]Jiang Ch G, Zhang X Y and Long Zh Ch, 2004.Study on activity of antivirus and mechanism of action of complex of prescription of Chinese herbal medicine on Transmissible Gastroenteritis of swine. Acta Veterinary et zootechnica sinica (姜成刚, 张秀英, 龙忠臣, 2004.中药复方制剂抗猪传染性胃肠炎病毒活性的研究.畜牧兽医学报, 35(5), 587-589)[19]Jiang Ch G, Zhang X Y and Long Zh Ch, 2005.Obersavation of activity of antivirus and mechanism of action of complex of prescription of Chinese herbal medicine on Transmissible Gastroenteritis of swine. Chinese Journal of Veterinary Medicine41（8）31-32 (姜成刚, 张秀英, 龙忠臣, 2005.中药抗猪传染性胃肠炎病毒活性及作用机理观察.中国兽医杂志41（8）31-32)[20]Liu X J, Zhang E H and Guo H L.2003.Review of study on Zhu shaqi. Journal of Shaanxi College of Traditional Chinese Medicine, 26(3)62-63(刘西京, 张恩户, 郭惠玲, 2003.朱砂七研究概况. 陕西中医学院学报, 26(3)62-63)[21]Lei Y D, Chen S S, Zhao X Y, Zhang L, Ma Y and Tong J M, 2012.Preparation and anti- TGEV activity of anthocyanin from purple sweet potato. Science and Technology of Food Industry, 33(21):349~352(雷用东, 陈姗姗, 赵晓燕, 张莉, 马越, 童军茂, 2012. 紫甘薯花色苷制备及其抗猪传染性胃肠炎病毒(TGEV)活性的初探.食品工业科技, 33:349~352)[22]Lei Y D, Wang D and Zhao X Y .2013.The Anthocyanin Profiles from Sambucus williamsii Hance and Analysis[23]Of Its Anti-Transmissible gastroenteritis virus (TGEV) Activity.Journal of Agricultural Biotechnology, 21(10):1185~1192(雷用东, 王丹, 赵晓燕, 2013.接骨木花色苷组成及抗猪传染性胃肠炎病毒(TGEV)活性分析.农业生物技术学报, 21(10):1185~1192)[24]Ren X F, Meng F D, Yin J Ch, Li G X, Li X L, Wang C h and G.Herrler, 2011. Action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus. PloS one6(5): e18669.[25]Rotman Y, M.Noureddin, J. J. Feld, J. Guedj, M. Witthaus, H. Han, Y. J. Park, S.-H. Park, T. Heller and M. G. Ghany, 2014. Effect of ribavirin on viral kinetics and liver gene expression in chronic hepatitis C. Gut, 63(1): 161-169.[26]Song X, Yin Zh Q, Li L, Cheng A Ch, Jia R Y, Xu J, Wang Y, Yao X P, Cheng L and Zhao X, 2013.Antiviral activity of sulfated Chuanminshen violaceum polysaccharide against duck enteritis virus in vitro .Antiviral Research 98: 344–351[27]Wei Z Y, Wang X B, Zhang H Y, Yang C H, Wang Y B, Xu D H, Chen H Y and Cui B A, 2011.Inhibitory effects of indigowoad root polysaccharides on porcine reproductive and respiratory syndrome virus replication in vitro. Antiviral Therapy, 16(3):357-63.[28]Wang X M, Li Z X and Ren L J.2010. The antioxidant effect research of Polygonum Cillinerve polysaccharide. Anhui Agriculture Sciences, 38( 34) :19346－19347．(王晓梅，李宗孝，任莉君, 2010.朱砂七多糖的抗氧化作用研究.安徽农业科学, 38( 34) :19346－19347)[29]Yang C W, Yang Y N, Liang P H, Chen C M, Chen W L, Chang H Y, Chao Y S.and Lee S J, 2007. Novel small-molecule inhibitors of transmissible gastroenteritis virus.Antimicrobial agents and chemotherapy 51(11): 3924-3931.[30]Zhan Y H.1994.Traditional Chinese Medicine Resource of Shennongjia.Wu han：Hubei Peoples, Publishing House, 410（詹亚华.1994.中国神农架中药资源.武汉:湖北人民出版社, 410）[31]Zhang W M, 2010.Study on antiviral and antibacterial activity of extract from Polygounm Taibaishanense and Polygonal Cillinerve [the doctoral dissertation] Yang Ling : Northwest A＆F University(张为民，2010.太白蓼和朱砂七提取物抗病毒及抑菌活性研究. [博士学位论文]杨凌:西北农林科技大学)[32]Zhang W M, Zhang Y M, Guo K K and Hu J, 2011.Active site selection of Polygounm Taibaishanense Kung extractives inhibiting bacterial and NDV in vitro. Journal of Chinese Veterinary medicine., 31( 5) :710－714(张为民，张彦明，郭抗抗，胡静, 2011.朱砂七提取物体外抑菌和抗病毒活性部位筛选.中国兽医学报, 31( 5) :710-714)[33]Zhao Q, Feng B P, Zhu X X, Wang H D and Yuan W H .2012. Study on the anti-HSV activity of total anthraquinone from Zhushaqi in vitro. Northwest Pharmaceutical Journal, 11, 27(6) 548-550(赵勤，冯宝平，朱萱萱, 王海丹，袁武会, 2012.朱砂七总蒽醌体外抗单纯疱疹病毒的研究.西北药学杂志, 11, 27(6) 548-550)[34]Zeuzem, S, I.M. Jacobson, T. Baykal, R. T. Marinho, F. Poordad, M. Bourlière, M. S. Sulkowski, H. Wedemeyer, E. Tam and P. Desmond .2014.Retreatment of HCV with ABT-450/r–ombitasvir and dasabuvir with ribavirin. New England Journal of Medicine.370(17): 1604-1614.[35]Zhou J F, Huang F, Hua X G, Cui L, Zhang W, ShenY, Yan Y J, Chen P, Ding D Zh and MouJ, 2010.Inhibition of porcine transmissible gastroenteritis virus (TGEV) replication in mini-pigs by shRNA.Virus research149(1): 51-55.[36]Zhao X N, Hu Y, Wang D, Guo L, Yang S, Fan Y, Zhao B, Wang Y and Abula S, 2011.Optimization of sulfated modification conditions of tremella polysaccharide and effects of modifiers on cellular infectivity of NDV. International journal of biological macromolecules, 49(1): 44-49
