海藻硫酸多糖和中药复方对鸡胚接种H9N2亚型禽流感病毒的作用
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摘要
为探讨海藻硫酸多糖(sulfated polysaccharide from algae,SPA)及中药复方提取液(compound extracts of Chinese medicine,CECM)对H9N2亚型禽流感病毒(avian influenza virus,AIV)的作用,将SPA及CECM以3种不同加药方式作用于接种了H9N2亚型AIV的10日龄SPF鸡胚中,无菌收集鸡胚尿囊液,测定血凝效价,检测2种药液对H9N2亚型AIV的预防、治疗及直接灭活作用。结果显示,SPA及CECM的最大安全浓度分别为50和200mg/mL;3种不同加药方式下,不同浓度SPA及CECM均能极显著降低鸡胚尿囊液中H9N2亚型AIV的血凝效价(P<0.01),以直接灭活作用组的血凝效价降低幅度最大,预防作用组和治疗作用组次之。预防作用组,CECM高剂量组(200mg/mL)的抗病毒效果显著优于SPA各剂量组(12.5、25和50mg/mL)及CECM低、中剂量组(50和100 mg/mL)(P<0.05);治疗作用组,SPA高剂量组(50 mg/mL)及CECM中、高剂量组(100和200mg/mL)抗病毒效果显著优于SPA低剂量组(12.5mg/mL)(P<0.05);直接灭活作用组,SPA及CECM的抗病毒效果均佳,CECM各剂量组的抗病毒效果与SPA各剂量组差异极显著(P<0.01)。结果表明,SPA和CECM对10日龄鸡胚几乎无毒副作用;SPA及CECM均能显著抑制H9N2亚型AIV在鸡胚尿囊液中的增殖,以直接灭活作用组的抗病毒效果最佳,其抗H9N2亚型AIV作用的强弱与药物浓度、用药和病毒的感染时间顺序密切相关。
To explore the effects of sulfated polysaccharide from algae(SPA)and the compound extracts of Chinese medicine(CECM)on H9 N2 subtype avian influenza virus(AIV),SPA and CECM were applied to the 10-day-old SPF chicken embryos inoculated with H9 N2 subtype AIV in 3 different ways.The chicken embryo allantoic fluid was collected aseptically,the blood coagulation titer was measured,and the prevention,treatment and direct inactivation of the two kinds of liquids to H9 N2 subtype AIV were tested.The results showed that the maximum safe concentrations of SPA and CECM were 50 and 200 mg/mL,respectively.Under different dosing methods,different concentrations of SPA and CECM could extremely significantly reduce the blood coagulation of H9 N2 subtype AIV in chicken embryo allantoic fluid(P<0.01).The hemocoagulation titer of the direct inactivation group decreased the most,the preventive group and the therapeutic group followed.In the preventive group,the anti-viral effect of CECM high dose group(200 mg/mL)was significantly better than that of SPA dose groups(12.5,25,50 mg/mL)and CECM low and medium dose groups(50,100 mg/mL)(P<0.05);SPA high-dose group(50 mg/mL)and CECM medium-and high-dose groups(100,200 mg/mL)were significantly better than SPA low group(12.5 mg/mL)(P<0.05);The direct inactivation group,SPA and CECM both had good antiviral effects,and the antiviral effects of CECM dose groups were extremely significantly different from those of SPA dose groups(P<0.01).The results showed that SPA and CECM had almost no toxic side effects on 10 days old chicken embryos;SPA and CECM could significantly inhibit the proliferation of H9 N2 subtype AIV in chicken embryo allantoic fluid,and the antiviral effect of direct inactivation group was the best.The strength of its anti-H9 N2 subtype AIV was closely related to the drug concentration,medication and viral infection time sequence.
To explore the effects of sulfated polysaccharide from algae(SPA)and the compound extracts of Chinese medicine(CECM)on H9 N2 subtype avian influenza virus(AIV),SPA and CECM were applied to the 10-day-old SPF chicken embryos inoculated with H9 N2 subtype AIV in 3 different ways.The chicken embryo allantoic fluid was collected aseptically,the blood coagulation titer was measured,and the prevention,treatment and direct inactivation of the two kinds of liquids to H9 N2 subtype AIV were tested.The results showed that the maximum safe concentrations of SPA and CECM were 50 and 200 mg/mL,respectively.Under different dosing methods,different concentrations of SPA and CECM could extremely significantly reduce the blood coagulation of H9 N2 subtype AIV in chicken embryo allantoic fluid(P<0.01).The hemocoagulation titer of the direct inactivation group decreased the most,the preventive group and the therapeutic group followed.In the preventive group,the anti-viral effect of CECM high dose group(200 mg/mL)was significantly better than that of SPA dose groups(12.5,25,50 mg/mL)and CECM low and medium dose groups(50,100 mg/mL)(P<0.05);SPA high-dose group(50 mg/mL)and CECM medium-and high-dose groups(100,200 mg/mL)were significantly better than SPA low group(12.5 mg/mL)(P<0.05);The direct inactivation group,SPA and CECM both had good antiviral effects,and the antiviral effects of CECM dose groups were extremely significantly different from those of SPA dose groups(P<0.01).The results showed that SPA and CECM had almost no toxic side effects on 10 days old chicken embryos;SPA and CECM could significantly inhibit the proliferation of H9 N2 subtype AIV in chicken embryo allantoic fluid,and the antiviral effect of direct inactivation group was the best.The strength of its anti-H9 N2 subtype AIV was closely related to the drug concentration,medication and viral infection time sequence.
引文
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[17]王长振,丛建波,先宏,等.SPA的分离纯化及其抗肿瘤作用研究[J].解放军药学学报,2010,26(4):283-286.WANG C Z,CONG J B,XIAN H,et al.Purification of sulfated polysaccharide from Laminaria japonica and studies of its anti-tumor mechanism[J].Pharmaceutical Journal of Chinese Peoples Liberation Army,2010,26(4):283-286.(in Chinese)
[18] BESEDNOVA N N,MAKARENKOVA I D,ZVYAGINTSEVA T N,et al.Antiviral activity and pathogenetic targets for seaweed sulfated polysaccharides in herpesvirus infections[J].Biochemistry Supplement,2016,10(1):31-42.
[19] AGUILAR-BRISENO J A,CRUZ-SUAREZ L E,SASSI J F,et al.Sulphated polysaccharides fromUlva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion,in NDV infection[J].Marine Drugs,2015,13(2):697-712.
[20]殷震,刘景华.动物病毒学[M].北京:科学出版社,1997.YIN Z,LIU J H.Animal Virology[M].Beijing:Science Press,1997.(in Chinese)
[21]郭鑫.动物免疫学实验教程[M].北京:中国农业大学出版社,2006.GUO X.Experimental Course of Animal Immunology[M].Beijing:China Agricultural University Press,2006.(in Chinese)
[22]陈晓霞,王林川.中草药抑制H5亚型禽流感病毒对鸡胚毒性的研究[J].广东畜牧兽医科技,2010,35(3):37-40.CHEN X X,WANG L C.The inhibition of five Chinese medicine herb preparations to lethal effects of H5subtype avian influenza virus to embryos[J].Animal Husbandry and Veterinary Science and Technology in Guangdong,2010,35(3):37-40.(in Chinese)
[23] PUJOL C A,RAY S,RAY B,et al.Antiviral activity against dengue virus of diverse classes of algal sulfated polysaccharides[J].International Journal of Biological Macromolecules,2012,51(4):412-416.
[24]吴彦霖,张媛,刘倩,等.鸡胚尿囊膜法评价板蓝根提取液抗H1N1流感病毒活性作用[J].科技导报,2016,34(13):78-82.WU Y L,ZHANG Y,LIU Q,et al.Evaluation of Radix Isatidis against H1N1influenza virus by using chicken embryo[J].Science&Technology Review,2016,34(13):78-82.(in Chinese)
[25]刘盛,陈万生,乔传卓,等.不同种质板蓝根和大青叶的抗甲型流感病毒作用[J].第二军医大学学报,2000,3:204-206.LIU S,CHEN W S,QIAO C Z,et al.Antiviral action of Radix Isatidis and Folium Isatidis from different germplasm against influenza A virus[J].Academic Journal of Second Military Medical University,2000,3:204-206.(in Chinese)
[26]刘晓婷,张沂,顾立刚,等.黄芩苷体外抗流感病毒作用的研究[J].天津中医药,2015,32(4):229-233.LIU X T,ZHANG Y,GU L G,et al.Study on the anti-influenza virus effect of baicalin in vitro[J].Tianjin Traditional Chinese Medicine,2015,32(4):229-233.(in Chinese)
[27]张瑞莉,华育平,刘娣,等.金银花提取液在鸡胚内抗新城疫病毒试验[J].中兽医医药杂志,2010,29(4):33-36.ZHANG R L,HUA Y P,LIU D,et al.Anti Newcastle disease virus test of honeysuckle extract in chicken embryo[J].Journal of Traditional Chinese Veterinary Medicine,2010,29(4):33-36.(in Chinese)
[28]霍晓青.抗新城疫病毒中药活性组分筛选的研究[D].保定:河北农业大学,2011.HUO X Q.Screening the active components in traditional Chinese medicine for anti-Newcastle disease virus[D].Baoding:Hebei Agricultural University,2011.(in Chinese)
[2] TONG X C,WENG S S,XUE F,et al.Human infection of novel avian influenza A(H7N4)virus[J].Journal of Infection,2018,77(3):249-257.
[3] BALZLI C,LAGER K,VINCENT A,et al.Susceptibility of swine to H5and H7low pathogenic avian influenza viruses[J].Influenza&Other Respiratory Viruses,2016,10(4):346-352.
[4] LI H,LIU X,CHEN F,et al.Avian influenza virus subtype H9N2affects intestinal microbiota,barrier structure injury,and inflammatory intestinal disease in the chicken ileum[J].Viruses,2018,10(5):E270.
[5]陈俊霞,孙鹏,许书珍,等.H9N2亚型禽流感病毒HA基因的表达及其单因子血清的制备[J].中国畜牧兽医,2015,42(11):2888-2894.CHEN J X,SUN P,XU S Z,et al.Expression of H9N2subtype avian influenza virus HA gene and preparation of its mono-specific serum[J].China Animal Husbandry&Veterinary Medicine,2015,42(11):2888-2894.(in Chinese)
[6]范建.禽流感H9N2病毒与H1N1流感病毒易发生重排[N].科技日报,2011-03-23(001).FAN J.Avian influenza H9N2virus and H1N1influenza virus are prone to rearrangement[N].Science and Technology Daily,2011-03-23(001).(in Chinese)
[7]辛朝安.禽病学[M].北京:中国农业出版社,2003.XIN C A.Avian Pathology[M].Beijing:China Agriculture Press,2003.(in Chinese)
[8] SEEKINGS A H,SLOMKA M J,RUSSELL C,et al.Direct evidence of H7N7avian influenza virus mutation from low to high virulence on a single poultry premises during an outbreak in free range chickens in the UK,2008[J].Infection,Genetics and Evolution,2018,64:13-31.
[9] HASSAN A O,AMEN O,SAYEDAHMED E E,et al.Adenovirus vector-based multi-epitope vaccine provides partial protection against H5,H7,and H9avian influenza viruses[J].PLoS One,2017,12(10):e0186244.
[10]王勇.禽流感的最新流行特点及防控措施[J].中国畜牧兽医文摘,2016,32(7):94.WANG Y.The latest epidemic characteristics and prevention and control measures of avian influenza[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine,2016,32(7):94.(in Chinese)
[11]贺丽茹,徐兆强.H9亚型低致病性禽流感的防控[J].兽医导刊,2011,5:73-74.HE L R,XU Z Q.Prevention and control of H9subtype low pathogenic avian influenza[J].Veterinary Orientation,2011,5:73-74.(in Chinese)
[12] SAMSON M,PIZZORNO A,ABED Y,et al.Influenza virus resistance to neuraminidase inhibitors[J].Antiviral Research,2013,98(2):174-185.
[13] TING L,PENG T.Traditional Chinese herbal medicine as a source of molecules with antiviral activity[J].Antiviral Research,2013,97:1-9.
[14]张薇,卢芳国,何迎春,等.麻杏石甘汤体外抗A型流感病毒作用的实验研究[J].实用预防医学,2007,5:1351-1353.ZHANG W,LU F G,HE Y C,et al.Experimental study on effect of Maxingshigan dectoctin on anti-influenza virus A in vitro[J].Practical Preventive Medicine,2007,5:1351-1353.(in Chinese)
[15]陈鹏举,解金辉,张光辉.自拟中药冬竹散对禽流感的治疗效果分析[J].中兽医学杂志,2017,1:71.CHEN P J,XIE J H,ZHANG G H.Analysis of therapeutic effect on avian influenza from Chinese traditional medicine Dongzhusan[J].Chinese Journal of Traditional Veterinary Science,2017 1:71.(in Chinese)
[16] SONG L,CHEN X,LIU X,et al.Characterization and comparison of the structural features,immune-modulatory and anti-avian influenza virus activities conferred by three algal sulfated polysaccharides[J].Marine Drugs,2016,14(1):4.
[17]王长振,丛建波,先宏,等.SPA的分离纯化及其抗肿瘤作用研究[J].解放军药学学报,2010,26(4):283-286.WANG C Z,CONG J B,XIAN H,et al.Purification of sulfated polysaccharide from Laminaria japonica and studies of its anti-tumor mechanism[J].Pharmaceutical Journal of Chinese Peoples Liberation Army,2010,26(4):283-286.(in Chinese)
[18] BESEDNOVA N N,MAKARENKOVA I D,ZVYAGINTSEVA T N,et al.Antiviral activity and pathogenetic targets for seaweed sulfated polysaccharides in herpesvirus infections[J].Biochemistry Supplement,2016,10(1):31-42.
[19] AGUILAR-BRISENO J A,CRUZ-SUAREZ L E,SASSI J F,et al.Sulphated polysaccharides fromUlva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion,in NDV infection[J].Marine Drugs,2015,13(2):697-712.
[20]殷震,刘景华.动物病毒学[M].北京:科学出版社,1997.YIN Z,LIU J H.Animal Virology[M].Beijing:Science Press,1997.(in Chinese)
[21]郭鑫.动物免疫学实验教程[M].北京:中国农业大学出版社,2006.GUO X.Experimental Course of Animal Immunology[M].Beijing:China Agricultural University Press,2006.(in Chinese)
[22]陈晓霞,王林川.中草药抑制H5亚型禽流感病毒对鸡胚毒性的研究[J].广东畜牧兽医科技,2010,35(3):37-40.CHEN X X,WANG L C.The inhibition of five Chinese medicine herb preparations to lethal effects of H5subtype avian influenza virus to embryos[J].Animal Husbandry and Veterinary Science and Technology in Guangdong,2010,35(3):37-40.(in Chinese)
[23] PUJOL C A,RAY S,RAY B,et al.Antiviral activity against dengue virus of diverse classes of algal sulfated polysaccharides[J].International Journal of Biological Macromolecules,2012,51(4):412-416.
[24]吴彦霖,张媛,刘倩,等.鸡胚尿囊膜法评价板蓝根提取液抗H1N1流感病毒活性作用[J].科技导报,2016,34(13):78-82.WU Y L,ZHANG Y,LIU Q,et al.Evaluation of Radix Isatidis against H1N1influenza virus by using chicken embryo[J].Science&Technology Review,2016,34(13):78-82.(in Chinese)
[25]刘盛,陈万生,乔传卓,等.不同种质板蓝根和大青叶的抗甲型流感病毒作用[J].第二军医大学学报,2000,3:204-206.LIU S,CHEN W S,QIAO C Z,et al.Antiviral action of Radix Isatidis and Folium Isatidis from different germplasm against influenza A virus[J].Academic Journal of Second Military Medical University,2000,3:204-206.(in Chinese)
[26]刘晓婷,张沂,顾立刚,等.黄芩苷体外抗流感病毒作用的研究[J].天津中医药,2015,32(4):229-233.LIU X T,ZHANG Y,GU L G,et al.Study on the anti-influenza virus effect of baicalin in vitro[J].Tianjin Traditional Chinese Medicine,2015,32(4):229-233.(in Chinese)
[27]张瑞莉,华育平,刘娣,等.金银花提取液在鸡胚内抗新城疫病毒试验[J].中兽医医药杂志,2010,29(4):33-36.ZHANG R L,HUA Y P,LIU D,et al.Anti Newcastle disease virus test of honeysuckle extract in chicken embryo[J].Journal of Traditional Chinese Veterinary Medicine,2010,29(4):33-36.(in Chinese)
[28]霍晓青.抗新城疫病毒中药活性组分筛选的研究[D].保定:河北农业大学,2011.HUO X Q.Screening the active components in traditional Chinese medicine for anti-Newcastle disease virus[D].Baoding:Hebei Agricultural University,2011.(in Chinese)