蓝耳病毒RX株的分离鉴定和中药体外抗病毒的作用研究
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摘要
本研究通过设计PRRSV PCR特异性引物,利用RT-PCR方法对广东地区某疑似暴发PRRS的猪场进行检测,结果为PRRSV阳性。然后用Marc-145细胞对发病猪肺脏、淋巴结进行病毒分离。从肺脏中分离得到一株病毒命名为PRRSV RX株。试验对分离病毒进行了鉴定,电镜观察结果可见胞浆内散布大量呈球形的病毒粒子,直径为40-60nm;病毒的TCID50为10-4.5/0. 1 ml。病毒为RNA病毒;RX株经56℃处理20-30min后丧失对细胞的感染力;病毒培养的最佳PH值是6. 8-7. 0。病毒动物回归试验表明,分离病毒接种40日龄的仔猪,临床表现体温升高和一定程度的呼吸症状。
选用黄芪、板蓝根、甘草、鱼腥草、山楂和青蒿六种中草药和为试验药物进行体外抗病毒试验。中草药采用传统的水煮浸提法分别提取并浓缩成100mg/m1溶液作为原液,高压灭菌处理备用。药物原液倍比稀释成不同浓度以测定其对Marc145细胞的毒性作用,来确定药物的安全浓度。以安全浓度为准,稀释成四个不同浓度在Marc145细胞上做抗病毒试验;采用三种作用方式:先加药物后加病毒,先加病毒后加药物,药物与病毒先作用一段时间后加入,以CPE、病毒滴度和MTT法测得的OD值为观测指标评价药物抗PRRSV病毒的活性,以探讨其抗病毒吸附、抗病毒复制或直接灭活病毒的作用方式。试验结果如下:
各药物对Marc145细胞的安全浓度分别为黄芪12.5 mg/mL、板蓝根3.125 mg/mL、甘草3.125 mg/mL、山楂3.125 mg/mL、鱼腥草1.526 mg/mL、青蒿1.526 mg/mL。
所筛选药物黄芪,板蓝根,甘草,山楂,鱼腥草,青蒿都具有抗病毒吸附作用而且保护作用均显著;先加病毒后加药物时,鱼腥草的作用比较稳定而板蓝根在3.125 mg/mL,1.562 mg/mL保护率最高。药物对病毒的杀灭作用时板蓝根在3.125 mg/mL,1.562 mg/mL的作用比较明显。不同药物对同种细胞的毒性不一样。
综合三种加药方式的效果考虑,板蓝根、青蒿能够抑制病毒,对细胞的保护率较高。
Isolation ,identification of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV) from Guangdong were performed in this study. One virus strain was successfully isolated from the tissues of pigs which were preliminarily diagnosed to be infected with PRRSV, designated RX. The isolate can proliferated on Marc-I45 cell line. After three passages, apparent CPE were observed in Marc-145 cell.Further experiments were done to identify the isolate. A large number of global particles (40-60nm in diameter) were observed under electron microscope. The TCIDS0 was 10-4.5/0. 1 ml. When the virus was treated at 56℃for 20-30 min ,the virus lost the ability to infect Marc-145 cell line. Slight improvement of body temperature and respiratory syndrome appeared when 40-day-old piglets were inoculated with the isolate. Above experiments proved the isolate to be PRRSV.
Six Chinese herbs such as Radix Astragali, Radix isatidis ,Radix Glycytthizae, Herba Houttuyniae ,Fructus Crataegi and Herba Artemisiae Annuae were used in anti-virus experiment in vitro. The Chinese herbs were Separately decocted and concentrated by heating to a final concentration of 100mg/ml respectively. All the drug-liquids were sterilized by autoclave and stored at 40C .In the Marc145ell culture experiment in vitro, the cytotoxic influence of different concentrations diluted at multiple proportions was studied to determine the highest non-toxic concentration, and anti-virus mechanism of the effective anti-PRRSVwas studied in three ways: adding medicine to the cultured Vero cell before adding virus, adding virus to the cultured Vero cell before adding medicine, and adding medicine and virus to the cultured Vero cell after they acted for a while. Antiviral activity against PRRSV was estimated in terms of Cytopathic effect, viral titre yields and OD values of MTT colorimetric method to determine the respective effects of anti-penetration activity against virus, restraint of virus propagation and direct destruction of virus , respectively. The results were as follows.
The highest non-toxic concentrations of medicaments were(mg/ml): Radix Astragali 12.5 mg/mL、Radix Glycytthizae3.125 mg/mL、Herba Houttuyniae 3.125 mg/mL、Fructus Crataegi 1.526 mg/mLand Herba Artemisiae Annuae1.526 mg/mL.
The best medicines in the three ways were screened out. Radix Glycytthizae and Herba Artemisiae Annuae provided good protection for cell. The protection rates of amantadine and neutral acriflavine were over 80% and this showed that they had better effect than Chinese herbal extracts.
选用黄芪、板蓝根、甘草、鱼腥草、山楂和青蒿六种中草药和为试验药物进行体外抗病毒试验。中草药采用传统的水煮浸提法分别提取并浓缩成100mg/m1溶液作为原液,高压灭菌处理备用。药物原液倍比稀释成不同浓度以测定其对Marc145细胞的毒性作用,来确定药物的安全浓度。以安全浓度为准,稀释成四个不同浓度在Marc145细胞上做抗病毒试验;采用三种作用方式:先加药物后加病毒,先加病毒后加药物,药物与病毒先作用一段时间后加入,以CPE、病毒滴度和MTT法测得的OD值为观测指标评价药物抗PRRSV病毒的活性,以探讨其抗病毒吸附、抗病毒复制或直接灭活病毒的作用方式。试验结果如下:
各药物对Marc145细胞的安全浓度分别为黄芪12.5 mg/mL、板蓝根3.125 mg/mL、甘草3.125 mg/mL、山楂3.125 mg/mL、鱼腥草1.526 mg/mL、青蒿1.526 mg/mL。
所筛选药物黄芪,板蓝根,甘草,山楂,鱼腥草,青蒿都具有抗病毒吸附作用而且保护作用均显著;先加病毒后加药物时,鱼腥草的作用比较稳定而板蓝根在3.125 mg/mL,1.562 mg/mL保护率最高。药物对病毒的杀灭作用时板蓝根在3.125 mg/mL,1.562 mg/mL的作用比较明显。不同药物对同种细胞的毒性不一样。
综合三种加药方式的效果考虑,板蓝根、青蒿能够抑制病毒,对细胞的保护率较高。
Isolation ,identification of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV) from Guangdong were performed in this study. One virus strain was successfully isolated from the tissues of pigs which were preliminarily diagnosed to be infected with PRRSV, designated RX. The isolate can proliferated on Marc-I45 cell line. After three passages, apparent CPE were observed in Marc-145 cell.Further experiments were done to identify the isolate. A large number of global particles (40-60nm in diameter) were observed under electron microscope. The TCIDS0 was 10-4.5/0. 1 ml. When the virus was treated at 56℃for 20-30 min ,the virus lost the ability to infect Marc-145 cell line. Slight improvement of body temperature and respiratory syndrome appeared when 40-day-old piglets were inoculated with the isolate. Above experiments proved the isolate to be PRRSV.
Six Chinese herbs such as Radix Astragali, Radix isatidis ,Radix Glycytthizae, Herba Houttuyniae ,Fructus Crataegi and Herba Artemisiae Annuae were used in anti-virus experiment in vitro. The Chinese herbs were Separately decocted and concentrated by heating to a final concentration of 100mg/ml respectively. All the drug-liquids were sterilized by autoclave and stored at 40C .In the Marc145ell culture experiment in vitro, the cytotoxic influence of different concentrations diluted at multiple proportions was studied to determine the highest non-toxic concentration, and anti-virus mechanism of the effective anti-PRRSVwas studied in three ways: adding medicine to the cultured Vero cell before adding virus, adding virus to the cultured Vero cell before adding medicine, and adding medicine and virus to the cultured Vero cell after they acted for a while. Antiviral activity against PRRSV was estimated in terms of Cytopathic effect, viral titre yields and OD values of MTT colorimetric method to determine the respective effects of anti-penetration activity against virus, restraint of virus propagation and direct destruction of virus , respectively. The results were as follows.
The highest non-toxic concentrations of medicaments were(mg/ml): Radix Astragali 12.5 mg/mL、Radix Glycytthizae3.125 mg/mL、Herba Houttuyniae 3.125 mg/mL、Fructus Crataegi 1.526 mg/mLand Herba Artemisiae Annuae1.526 mg/mL.
The best medicines in the three ways were screened out. Radix Glycytthizae and Herba Artemisiae Annuae provided good protection for cell. The protection rates of amantadine and neutral acriflavine were over 80% and this showed that they had better effect than Chinese herbal extracts.
引文
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