肉食动物细小病毒的检测方法

从猫、犬、貂、貉、兰狐和豹等多种肉食动物中分离到许多类似的细小病毒,并以其宿主分别称为:猫泛白细胞减少症病毒(FPLV)、犬细小病毒(CPV)、水貂肠炎病毒(MEV)、貉细小病毒(RPV)、兰狐细小病毒(BFPV)和豹细小病毒(LPV),这些细小病毒均具有细小病毒科及细小病毒属的典型特点,在形态、理化特性上极为相似,本文仅就上述几种细小病毒的检测方法做一简述,供科学研究、临床实践、海关检疫等有关人员参考借鉴。     

猫细小病毒NS部分基因的克隆及序列分析

将疫苗用猫细小病毒(FPV)株提取基因组DNA,针对NS特定片段设计引物,利用PCR扩增出了部分基因并将该基因克隆到pMD18-T Vector中测序.结果表明,该基因长613bp,编码204个氨基酸.分离株基因与犬的细小病毒(CPV)、貂的细小病毒(MEV)毒株核苷酸同源性均为99%.氨基酸同源性达到97%、98%以上,与犬、貂的肠炎病毒有密切的亲缘关系.     

肉食兽细小病毒通用PCR诊断技术的建立

根据肉食兽细小病毒核苷酸序列高度同源的特点，设计合成了1对引物，以犬细小病毒、貂肠炎病毒和猫泛和白细胞减少症病细胞培养物为DNA模板，进行PCR特异性片段扩增，扩增片段大小为0．6kb。结果表明，扩增产物与设计的2个引物之间的序列大小一致。通过通用性、特异性与敏感性试验及对临床检样品检测，证明本法对肉食兽细小病毒通用。且具有快速、特异和高度敏感的特点。     

水貂肠炎病毒、猫泛白细胞减少症病毒以及犬细小病毒2型间的关系比较

水貂肠炎病毒(MEV)、猫泛白细胞减少症病毒(FPV)、犬细小病毒2型(CPV-2)为隶属于细小病毒属的三种极为相似的自主复制型病毒。最初人们主要根据水貂、猫、犬患病症状相似的特点,注意到它们间可能的密切关系。时至今日,已对这3种病毒的许多方面进行了分析研究。所有试     

番鸭细小病毒与鹅细小病毒PCR鉴别诊断方法的建立

根据番鸭细小病毒（MDPV）和鹅细小病毒（GPV）基因组的非同源序列各设计了1对引物MDPVF1／R1和GPVF1／R1，建立了一种PCR方法，用该PCR方法分别对GPV、MDPV、鸭瘟病毒（DPV）、鸭肝炎病毒（DHV）、鸭呼肠孤病毒（DRV）、犬细小病毒（CPV）和猫泛白细胞减少症病毒（FPLV）的病毒培养物及其核酸进行扩增。结果，引物MDPVF1／R1仅特异性扩增出MDPV的900bp核酸片段，引物GPVF1／R1仅特异性扩增出GPV的465bp核酸片段。表明，建立的PCR方法可用于GPV和MDPV的鉴别诊断。     

犬细小病毒四川株的分离与鉴定

为更好地了解我国细小病毒流行情况,笔者从四川农大动物医院采集5份患出血性肠炎犬的粪便,通过猫肾细胞(F81)传代培养后,成功分离到两株犬细小病毒。对两株病毒进行病毒形态学、理化学、血凝试验、PCR鉴定与分子生物学系统鉴定后,证实分离株是犬细小病毒,通过VP2结构蛋白测序分析表明,新分离到的两株细小病毒抗原型分别属于CPV-2a和CPV-2b。     

浅谈犬细小病毒酌防治

1病原 犬细小病毒于细小病毒科,细小病毒属,犬细小病毒（Canine Parovirus,CPV）该病毒粒子细小,直径为20μm,无囊膜,单股DNA。对新生组织细胞有亲和力,可以在犬肾细胞及猫肾细胞上生长繁殖。     

犬细小病毒病的病原学研究进展

犬细小病毒病是由犬细小病毒感染引起的一种急性传染病,以剧烈的呕吐、出血性肠炎和白细胞显著减少为主要特征,并可引起犬急性心肌炎。症状与猫泛白细胞减少症相似,是危害我国养犬业最为严重的传染病之一,可造成严重的经济损失。文章从病毒生物学特性、基因组结构、疫苗开发研制以及病原的检测方法等角度对犬细小病毒病的病原学研究进行了综述。     

浅谈犬细小病毒的防治

1病原 犬细小病毒于细小病毒科，细小病毒属，犬细小病毒（Canine Parovirus，CPV）该病毒粒子细小，直径为20μm，无囊膜，单股DNA。对新生组织细胞有亲和力，可以在犬肾细胞及猫肾细胞上生长繁殖。     

犬细小病毒四川株的分离与鉴定

采集5份患出血性肠炎犬的粪便,通过猫肾细胞(F81)传代培养后,成功分离到2株病毒。对2株病毒进行病毒形态学、理化学、血凝试验、PCR鉴定与分子生物学系统鉴定后,证实分离株是犬细小病毒,通过VP2结构蛋白测序分析表明,新分离到的2株细小病毒抗原型分别属于CPV-2a和CPV-2b。     

Virus inactivation by ethylene oxide containing gases.

Gases containing ethylene oxide mixed with carbon dioxide alone (Etox®) or together with methyl formate (Etoxiat®) were employed for virus treatment in a way that has been shown efficient for the killing of bacteria. A number of viruses selected for their capacity to withstand chemical or physical treatments were tested under varying conditions, including in a dried state in the presence of high amounts of organic matter (animal spillings). The viruses tested were enteroviruses, paramyxovirus (NDV), poxvirus and parvovirus, and they were all inactivated to a high degree.     

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.     

Virus survival in slurry: analysis of the stability of foot-and-mouth disease, classical swine fever, bovine viral diarrhoea and swine influenza viruses

Farm slurry can be highly contaminated with viral pathogens. The survival of these pathogens within slurry is important since this material is often distributed onto farm land either directly or after heat treatment. There is clearly some risk of spreading pathogens in the early stages of an outbreak of disease before it has been recognized. The survival of foot-and-mouth disease virus, classical swine fever virus, bovine viral diarrhoea virus and swine influenza virus, which belong to three different RNA virus families plus porcine parvovirus (a DNA virus) was examined under controlled conditions. For each RNA virus, the virus survival in farm slurry under anaerobic conditions was short (generally ≤ 1 h) when heated (to 55°C) but each of these viruses could retain infectivity at cool temperatures (5°C) for many weeks. The porcine parvovirus survived considerably longer than each of the RNA viruses under all conditions tested. The implications for disease spread are discussed.     

伪狂犬病毒单克隆抗体的制备和鉴定

以伪狂犬病毒（PRV）作为免疫原,免疫BALB／c小鼠,融合之后经间接ELISA、IFA和IHC试验进行杂交瘤筛选,共获得2株能分泌针对PRV单克隆抗体（简称单抗）的杂交瘤细胞株。2株猪伪狂犬病毒单抗与猪细小病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒均无交叉反应,IFA结果显示单抗能与接种于猪睾丸细胞（ST）的PRV发生特异性反应,IHC结果显示单抗能用感染了PRV的猪神经组织发生反应,证实抗PRV单克隆抗体具有良好的特异性和敏感性,此结论为猪伪狂犬病毒抗原表位分析及相关抗原抗体诊断试剂盒的研制奠定了基础。     

Establishment of a Duplex PCR Assay for Detection of Duck Newcastle Disease Virus and Muscovy Duck Parvovirus

According to the gene sequences of duck Newcastle disease virus (NDV) L gene and Muscovy duck parvovirus (MDPV) Vp3 gene in GenBank, two sets of specific primers were designed by Primer Premier 5.0. The duplex PCR assay was developed through optimization of reaction conditions and validation of specificity, sensitivity and repetitiveness of the method. The sensitivity of the assay was 30 fg for NDV and 16 fg for MDPV. No specific bands of the same sizes were amplified from other duck pathogens, such as duck plague virus, duck hepatitis virus, duck circovirus, H9 subtype avian influenza virus, duck flavivirus, Salmonella, E.coli, avian Pasteurella multocida. This duplex PCR assay was a quick, sensitive and specific test for detection of duck NDV and MDPV, and would be useful for the control of these viruses in ducks.     

水貂阿留申病毒的分子生物学研究进展

从水貂阿留申病毒(ADV)基因组特点出发,就阿留申病毒的分子生物学研究进展作以简单综述。     

猪细小病毒、猪伪狂犬病病毒和猪圆环病毒2型多重PCR检测方法的建立与应用

根据GenBank中已发表的猪细小病毒(porcine parvovirus,PPV)、猪伪狂犬病病毒(pseudorabies virus,PRV)和猪圆环病毒2型 (porcine circovirus type 2,PCV2)基因序列,对各病毒基因区进行同源性分析,确定PPV 的VP2、PRV的 gD、和PCV2的ORF2基因为各病毒的诊断靶序列,设计特异性引物,在建立各病毒单项PCR技术的基础上,优化多重PCR反应条件,建立了3种病毒的多重PCR技术,可同时扩增PPV 313 bp、 PRV 217 bp和PCV2 447 bp的特异性片段。用多重PCR技术与单项PCR技术对比检测试验证明两者的符合率为100％,表明建立的多重PCR检测方法,具有特异、快速、准确的特点,可同时鉴别诊断这3种病毒。从10个发病猪场和门诊病例的病猪采集的211份样品,用建立的多重PCR检测方法,检出PPV阳性42份,阳性率为19.91%;PRV阳性26份,阳性率为12.32%;PCV2阳性56份,阳性率为26.54%;2种以上病毒混合感染25份,混合感染阳性率为11.85%。检测结果表明,山西省猪群已感染这3种疫病。     

新城疫、禽流感病毒同胚接种的研究

根据新城疫病毒、禽流感病毒两种病毒均能在鸡胚中繁殖的特点,将两种病毒以最适稀释比例稀释后,分别接种于9—11日龄的鸡胚尿囊腔,同时进行两种病毒同胚培养,并利用血凝试验进行病毒效价的测定。结果表明,在同一鸡胚中两种病毒均能较好地增殖,病毒间干扰现象不明显。本试验在增强疫苗的免疫原性和进一步阐明病毒的增殖机理方面具有重要意义。     

Establishment of Indirect ELISA Detection Method for Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease of pigs. Porcine epidemic diarrhea virus (PEDV) is the causative agent of PED. PED has caused significant economic losses to the pig industry. In this study,the purified PEDV as the coating antigen, by optimizing the ELISA reaction conditions,the indirect ELISA antibody detection method was established. The optimized reaction conditions were as follows: Antigen working concentration was 20 μg/mL; Serum sample dilution was 1:500;It was coated at 4℃ overnight; The plates were blocked by 5% calf serum incubated at 37℃ for 1 h; The secondary antibody was diluted at 1:10 000,incubated at 37℃ for 1 h. It was judged as positive when the cutoff value D_{450 nm}≥0.289,as negative when D_{450 nm}≤0.236,and as suspicious between 0.289 and 0.236.It could not react with the positive sear of other six viruses such as porcine respiratory and reproductive syndrome virus,porcine circovirus 2, classical swine fever virus, porcine parvovirus,pseudo rabies virus and foot-and-mouth disease virus. The variation coefficient of repeated test was less than 10%.74 pig serum samples from Jiangsu, Jiangxi, Fujian and Guangdong were detected,and the positive rate was 84%.It indicated that this method could be used for PEDV epidemiological surveys and diagnosis in the future.     

猪繁殖与呼吸综合症病毒单克隆抗体的制备和鉴定

以猪繁殖与呼吸综合征病毒（PRRSV）作为免疫原,免疫BALB／c小鼠,经间接ELISA、IPMA和IFA试验进行杂交瘤筛选,共获得2株能分泌针对PRRSV单克隆抗体（简称单抗）的杂交瘤细胞株,分别命名为3D10和4H11,3D10亚类为IgG1,4H11亚类为IgG2b,单抗腹水的间接ELISA效价均达到1．0×10^7,染色体数目介于90～110之间。2株单抗与猪细小病毒、猪伪狂犬病毒、猪圆环病毒2型均无交叉反应,IPMA和IFA结果显示单抗均能与接种于猴肾细胞（Marc145）的PRRSV发生特异性反应,证实抗PRRSV单抗具有良好的特异性和敏感性,为PRRSV抗原表位分析及相关抗原抗体诊断试剂盒的研制奠定了基础。