采用实时定量PCR技术评价三种药物体外抗鸭瘟病毒效果

采用实时定量PCR技术,对阿昔洛韦、利巴韦林及禽重组干扰素等三种药物体外抗鸭瘟病毒的作用进行了评价。结果显示,阿昔洛韦能有效抑制鸭瘟病毒在鸭胚成纤维细胞中的增殖。研究结果为抗鸭瘟病毒药物筛选提供了一种有效的技术平台,同时提示阿昔洛韦制剂有可能作为一种有效的抗鸭瘟病毒药物用于其感染的防治。     

探针实时荧光定量PCR检测白藜芦醇体外抗鸭瘟病毒活性方法的建立及应用

建立以TaqMan荧光探针为特点的PCR,检测白藜芦醇体外抗鸭瘟病毒活性。本研究针对鸭瘟病毒UL30基因序列设计特异性引物和TaqMan荧光探针,建立鸭瘟病毒TaqMan探针实时荧光定量PCR检测方法,并验证方法的特异度、敏感度和稳定性及对临床样品进行检测;运用该方法检测白藜芦醇对鸭胚成纤维细胞接种鸭瘟病毒后细胞中病毒增殖情况的影响,用以评价该药物体外抗鸭瘟病毒效果。结果显示,该方法建立的定量标准曲线阈值循环数（Threshold cycle,Ct）与模板拷贝数呈良好线性关系（R2=0.997）,斜率为-3.328,扩增效率（E）为99.7%,具有良好的特异度、敏感度和稳定性并能对临床样本进行准确的检测。运用该方法检测白藜芦醇体外抗鸭瘟病毒结果显示：经白藜芦醇处理后,染毒细胞中病毒拷贝数为（7.71±0.37）×10^4,与病毒对照组比较（5.63±0.26）×10^6明显减少（P〈0.01）。结果表明,TaqMan探针实时荧光定量PCR方法建立并成功用于体外药物抗鸭瘟病毒的检测;白藜芦醇具有良好的体外抗鸭瘟病毒活性。     

外源病毒检验用抗鸭瘟病毒特异性阳性血清的制备与检定

为制备鸭瘟种毒和鸭瘟活疫苗检验用抗鸭瘟病毒特异性阳性血清,对6个月龄左右的山羊进行五次免疫。最后一次免疫后2周采血并分离血清,血清经混合、分装、冷冻真空干燥后,对其进行了无菌检验、外源病毒检验、剩余水分测定、中和效价测定、均匀性检验。结果表明,本研究制备的抗鸭瘟病毒阳性血清特异性良好,无菌检验、外源病毒检验和剩余水分测定均符合《中华人民共和国兽药典》(2010年版三部)规定;血清中和效价大于1∶100,均匀性良好,可满足兽药典标准中鸭瘟种毒和鸭瘟活疫苗的鉴别检验和外源病毒检验。     

鸭瘟病毒河南株gG基因的克隆与同源比较

鸭瘟是由鸭瘟病毒(Duck plague virus,DPV)引起的鸭、鹅和其他雁形目禽类的一种急性、热性、败血性传染病[1]。该病以流行广泛、传播迅速、发病率和死亡率高为特征。鸭瘟病毒的研究起步较晚,其分子生物学方面的研究相对滞后。试验以鸭瘟病毒河南     

体外抗鸭坦布苏病毒的药物筛选试验

为了探讨抗病毒药物对鸭坦布苏病毒的作用,选用5种常用的抗病毒药物(金刚乙胺、脱氧若卡素钠、病毒灵、利巴韦林、阿昔洛韦)进行体外抗鸭坦布苏病毒的药物筛选试验。结果表明,利巴韦林药物浓度为0.156 mg/m L~0.625 mg/m L时,在DF-1细胞中能抑制鸭坦布苏病毒的增殖、利巴韦林在体外对鸭坦布苏病毒具有抗病毒作用;而阿昔洛韦、金刚乙胺、脱氧若卡素钠、病毒灵等4种药物对鸭坦布苏病毒无明显抗病毒作用。     

鸭瘟病毒转移质粒的构建

为了建立重组鸭瘟病毒技术,构建了鸭瘟病毒转移质粒。在对鸭瘟强毒和弱毒株TK基因进行测序分析后,将鸭瘟病毒TK-UL24DNA片段克隆于pUC18载体中,构建了质粒pTK;将PCR扩增的GFP真核表达盒插入pTK质粒的TK基因内部,获得转移载体质粒pTK-GFP。鸭瘟病毒TK-UL24测序分析表明鸭瘟强、弱毒株TK基因序列完全相同;转移载体携带Pcmv-GFP-SV40pA表达盒,测序验证其序列与源序列一致。pTK-GFP在脂质体介导下,转染鸭胚成纤维细胞和鸭肾细胞,在荧光显微镜下观察绿色荧光蛋白表达情况。质粒转染细胞后,绿色荧光蛋白得到了有效的表达,为进一步开展重组鸭瘟病毒的研究和构建具有遗传标记的鸭瘟疫苗奠定了基础。     

鸭瘟病毒PCR快速检测方法的建立

根据Gen Bank公布的鸭瘟病毒UL30基因保守序列设计了一对引物,建立了检测鸭瘟病毒PCR方法,并对其特异性、敏感性进行了研究。该PCR方法对鸭瘟病毒扩增结果为阳性,对照毒株扩增结果均为阴性;对鸭瘟病毒检测的灵敏性为1 pg总DNA量。以上结果表明该PCR方法特异性强、敏感性高、简便、快速,可用于鸭瘟的早期确诊和病毒鉴定。     

"双黄"在鸭胚中抗鸭瘟病毒的效力试验

用双黄杀毒退烧颗粒剂(简称“双黄”)对8组鸭胚予以不同剂量的抗鸭瘟病毒效力试验,结果表明其在鸭胚中能抑制鸭瘟病毒增殖。     

野生水禽鸭瘟病毒的分离与鉴定

取急性死亡的某动物园野生水禽进行病毒分离,通过鸡(鸭)胚接种、动物回归试验、病毒主要理化特性测定、免疫预防试验等证实分离株为鸭瘟病毒,揭示野生水禽完全有自然感染鸭瘟的可能性,家鸭与野生水禽在相互间传播鸭瘟病毒上具有双向性.     

一株鸭瘟病毒(GM株)的分离与鉴定

针对山东潍坊地区鸭瘟零星发病现象,从潍坊采集具有典型症状病料,通过鸭胚接种,分离出一株病毒。通过分子生物学以及阳性血清定性中和试验鉴定为鸭瘟病毒(DPV),命名为鸭瘟病毒GM株。结果显示,该本病毒可适应鸭胚和鸡胚成纤维细胞;鸭胚ELD50为10-5.5/0.2 m L;该病毒无血凝活性,不能凝集1%鸡红细胞;本动物回归,使樱桃谷鸭在5~6 d死亡,成功复制出鸭瘟病毒;鸭瘟病毒GM株与鸭瘟病毒鸡胚化弱毒株(CVCC AV1222)疫苗的免疫攻毒试验表明,传统鸭瘟疫苗对该分离株具有保护力。初步确定该病毒为传统鸭瘟野毒。     

鸭痘病毒TaqMan荧光定量PCR检测方法的建立

为建立检测鸭痘病毒的TaqMan荧光定量PCR方法,本试验克隆了鸭痘病毒P4b基因,构建重组质粒pMD-DPV-P4b,并将其作为标准阳性模板。参照GenBank收录的禽痘病毒P4b基因设计合成1对特异性引物及与该引物相匹配的特异探针。以定量的10倍系列稀释的质粒pMD-DPV-P4b为标准品,通过对反应条件进行优化,建立了一种检测鸭痘病毒的TaqMan荧光定量PCR方法。结果显示,该方法与禽流感病毒、鸭黄病毒、鸭肝炎病毒、新城疫病毒、鸭瘟病毒和小鹅瘟病毒等其他水禽病毒,以及山羊痘病毒和鸡痘病毒等其他痘病毒均无交叉反应,特异性好。该方法最低检测限为1.29×10²拷贝/μL,比普通PCR检测方法高100倍。组内和组间变异系数均小于2%。结果表明,本试验所建立方法具有灵敏、特异、安全、快速的特点,适用于鸭痘病毒的检测。     

Establishment of TaqMan Real-time PCR Method for Detection of Duck Poxvirus

To establishment a TaqMan Real-time PCR method for detection of duck poxvirus (DPV),we cloned the P4b gene of DPV.The specific primers and probe were designed according to the nucleotide sequence of avipoxvirus available in GenBank.Recombinant plasmid pMD-DPV-P4b was employed as positive standard template for Real-time PCR.By optimization of reaction conditions,a TaqMan Real-time PCR method for detection of DPV was established.The results of specificity test proved this method had no cross-react with other waterfowl vial agents and poxviruses including avian influenza virus,duck flavivirus,duck hepatitis virus,Newcastle disease virus,duck entertitis virus,goose parvovirus,goatpox virus and fowlpox virus.The detection limit of the assay was 1.29×10² copies/μL of viral DNA,which was 100 times higher than that of the routine PCR.Reproducibility test showed that the CVs of intra assay and inter assay were both less than 2%.Above results supported that the assay was suitable for the detection of DPV very well.     

鸭坦布苏病毒和鸭瘟病毒二重荧光定量RT-PCR方法的建立

The study was conducted to establish the duplex Real-time PCR assay for detecting both duck tembusu virus (DTMUV) and duck plague virus (DPV). According to the sequences of DTMUV E gene and DPV UL6 gene in GenBank, two sets of specific oligonucleotide primers for DTMUV and DPV along with two TaqMan probes were designed. The duplex Real-time PCR assay was developed through optimization of reaction conditions and validation of specificity, sensitivity and repetitiveness of the method. The sensitivity of the assay were both 100 template copies for DTMUV and DPV. There was no specific bands of the same sizes were amplified from other duck pathogens, such as duck Newcastle disease virus, duck hepatitis virus, muscovy duck parvovirus, duck circovirus, H9 subtype avian influenza virus, egg drop syndrome virus. This duplex Real-time RT-PCR assay is a sensitive, quick, specific and quantitative test for detection of DTMUV and DPV, and will be useful for the control of these viruses in ducks.     

Screening of the Drugs Against Feline Infectious Rhinotracheitis Virus in vitro

This study was aimed to detect the efficacy of antiviral drugs against feline infectious rhinotracheitis virus. F81 cell was used to construct a model for drug screening against feline infectious rhinotracheitis virus in vitro. Virus inhibition rate was detected with MTT assay and calculated with SPSS software. The IC₅₀ of acyclovir, ribavirin, L-lysine, isatis root and Astragalus polysaccharides were 9.5,3.3,3.4,161.0 and 4.7 μg/mL, respectively,the IC₅₀ of polyinosinic was 6.0 mg/mL. The TI of them were 76.8, 39.3, 2 588.0, 4.5, 78.7 and 5.8, respectively. The effect of L-lysine and Astragalus polysaccharides were most significant.     

抗猫传染性鼻气管炎病毒药物的体外筛选

试验旨在检测抗病毒药物对猫传染性鼻气管炎病毒的有效性。利用F81细胞建立抗猫传染性鼻气管炎病毒药物的体外筛选模型,采用MTT法检测,计算病毒的抑制率。结果显示,阿昔洛韦、利巴韦林、L-赖氨酸、板蓝根和黄芪多糖的半数有效浓度（IC₅₀）分别为9.5、3.3、3.4、161.0和4.7 μg/mL,聚肌胞IC₅₀为6.0 mg/mL,治疗指数TI分别为76.8、39.3、2 588.0、4.5、78.7和5.8。结果表明,L-赖氨酸和黄芪多糖为高效抗猫传染性鼻气管炎病毒药物。     

Rapid diagnosis of duck plagues virus infection by loop-mediated isothermal amplification

Duck virus enteritis is a serious disease among farmed and free-living ducks (Anatidae) and a constant threat to the commercial duck industry in China. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to rapidly detect and diagnose duck plague virus (DPV) in both farmed and wild waterfowl, and compared with polymerase chain reaction (PCR) method and real-time PCR method in accuracy, sensitivity and specificity. A set of four specific primers was successfully designed to recognize six distinct genomic sequences of UL6 protein from DPV, including one forward inner primer, one back inner primer and two outer primers. The optimum reaction temperature and time were verified to be 61.5 °C and 60 min, respectively. Comparative experiments showed that LAMP assay was a simple, rapid, accurate, sensitive and specific method for detecting DPV, and was superior to PCR assay in sensitivity and specificity for DNA amplification. In addition, challenge tests indicated the newly developed LAMP method was more sensitive for the diagnosis of DPV infection than virus isolation and PCR. LAMP assay would be a good alternative method for on-farm disease diagnosis.     

二重PCR检测鸭瘟病毒和鹅细小病毒的初步研究

根据鸭瘟病毒(Duck plague virus,DPV)DNA聚合酶基因和鹅细小病毒(Goose parvovirus,GPV)NS基因序列分别设计2对引物,应用这2对引物对混合样品中鸭瘟病毒和鹅细小病毒进行了二重PCR扩增。PCR产物的琼脂糖凝胶电泳结果表明2条特异性带为563bp(DPV)和1146bp(GPV),与实验设计相符,且与常规单一PCR结果一致。二重PCR反应检测出的DPV含量相当于20个PFU;检测出的GPV含量相当于0.1个ELD50,对禽痘病毒和减蛋综合征病毒的核酸未能扩增出任何条带,特异性良好。该二重PCR能够在一次扩增反应中检测两种病毒,为两种鹅病的病原检测和诊断提供了一种简便、经济、快速的手段。     

Pharmacokinetics of acyclovir after single intravenous and oral administration to adult horses

The purpose of the study reported here was to describe the bioavailability and pharmacokinetics of acyclovir after intravenous and oral administration to horses. Six healthy adult horses were used in a randomized cross-over study with a 3 x 3 Latin square design. Three treatments were administered to each horse: 10 mg of injectable acyclovir/kg of body weight in 1 L of normal saline delivered as an infusion over 15 minutes; 10 mg of acyclovir/kg in tablets by nasogastric intubation; and 20 mg of acyclovir/kg in tablets by nasogastric intubation. A 2-week washout period was provided between each treatment. Serum samples were obtained for acyclovir assay using reversed-phase, high-performance liquid chromatography with fluorescence detection. Deproteinated serum was injected onto a C18 column, and elution occurred under isocratic conditions. The limit of quantification was 0.04 microg/mL. The assay exhibited suitable accuracy, precision, and recovery. The IV data were analyzed by a 3-compartment model, and oral data were analyzed noncompartmentally. Intragastric acyclovir administration at either dose was associated with high variability in serum acyclovir-time profiles, low Cmax, and poor bioavailability. The dosage of 20 mg/kg was associated with mean (+/- SD) Cmax of 0.19 +/- 0.10 microg/mL, and bioavailability was 2.8%. Inhibition of equine herpesvirus has been reported to require significantly higher acyclovir concentrations than those obtained here. The results of this study do not support a therapeutic benefit for the oral administration of acyclovir up to doses of 20 mg/kg.     

鸭瘟病毒UL35基因PCR方法的建立与初步应用

本研究建立了检测鸭瘟病毒（Duck pl ague vi rus,DPV）的PCR方法,并运用建立的检测方法对分离毒株和人工感染样品进行临床应用检测。根据GenBank（登录号为EF643558）中的DPV UL35基因保守区域,设计合成了一对引物,以DPV疫苗株为模板,优化PCR反应条件,建立了一种快速、有效的DPVPCR方法。结果显示：该方法能从DPV中扩增到与预期大小相符,长度为354 bp的特异性片段,而对禽流感病毒（AIV）、番鸭呼肠孤病毒（MDRV）、新城疫病毒（NDV）、番鸭细小病毒（MPV）、鹅细小病毒（GPV）等样品的扩增结果均为阴性;检测灵敏度达到470 ng病毒DNA。应用该方法对3株DPV分离株和6份由DPV BL8毒株人工感染鸭的肝脏和脾脏等组织进行PCR检测均为阳性。表明所建立的DPV PCR方法特异性强、灵敏度高,可用于DPV的临床诊断和流行病学调查。