S2基因失活突变马传染性贫血病毒感染性克隆的构建及感染性研究

为研制马传染性贫血病毒(EIAV)标记疫苗株,本研究在EIAV驴胎皮肤(FDD)细胞弱毒疫苗株感染性克隆pEIAVFDDV3-8的S2基因内部引入两个终止密码子,致使S2基因终止表达,构建了一株S2基因突变的感染性克隆pEIAVFDDV3-8△S2。用该重组质粒转染FDD细胞,盲传至第4代时,转染细胞病变明显,电镜下可见细胞内的典型病毒颗粒。反转录酶活性检测和Real-time PCR对病毒拷贝数的检测均证明,获得了EIAVS2基因终止表达的感染性克隆毒株,命名为EIAVFDDV3-8△S2。     

逆向遗传学技术在猪瘟病毒研究中的应用

猪瘟是猪的最严重疾病之一,其病原 是猪瘟病毒,基因组为单股正链RNA。猪瘟 弱毒疫苗在猪瘟的免疫防治中发挥了巨大作 用,但由于弱毒疫苗免疫的动物不能从血清 学上与自然感染动物区分,使其应用受到很 大限制,研制标记疫苗是解决这个问题的重 要途径。由于RAN的结构不稳定成为阻碍 RNA病毒研究的主要障碍,所以病毒分子生 物学是新型猪瘟疫苗研究的必要手段。近年 来兴起的逆向遗传研究将RNA病毒的基因 组转化为cDNA,文章就猪瘟病毒逆向遗传 研究的意义、方法、概况及其在猪瘟新型疫苗 研究中的应用进行了全面综述。     

1株高致病性血凝性脑脊髓炎病毒的分离与鉴定

利用细胞培养方法从临床表现神经症状的死亡仔猪脑组织内分离获得1株病毒,同时对该病毒进行电镜负染观察、昆明种小鼠致病性试验、RT-PCR检测和部分基因测序分析。电镜观察可见细胞培养物中有大量的直径约115 nm的冠状病毒样粒子;接种病毒的小鼠均出现典型的神经症状,死亡率100%;血凝性脑脊髓炎病毒(HEV)特异性引物RT-PCR扩增结果阳性;其S基因序列与GenBank中登录的HEV-67 N相应基因序列的同源性高达99.6%。结果表明,分离的病毒为猪血凝性脑脊髓炎病毒,分离株暂命名为HEV-CC-2007。     

Association between bovine-leukosis virus seroprevalence and herd-level productivity on US dairy farms

Bovine-leukosis virus (BLV; also termed ‘bovine-leukemia virus’) is a retrovirus that primarily affects lymphoid tissue of dairy and beef cattle. Our objective was to investigate the association between BLV infection and annual value of production (AVP) on dairy herds within the United States, as part of the USDA National Animal Health Monitoring System’s 1996 dairy study. 1006 herds (in 20 states) with at least 30 dairy cows were interviewed during 1996. The agar-gel immunodiffusion test was used to detect serum antibodies to BLV. 10–40 cows from each herd were tested and each tested cow was classified as negative or positive based on results of a single test.

A multivariable regression model was used with the 976 herds with complete data for analysis. When compared to herds with no test-positive cows, herds with test-positive cows produced 218 kg per cow (i.e. 3%) less milk. The average reduction in AVP was $ 59 per cow for test-positive herds relative to test-negative herds. For the dairy industry as a whole, BLV seropositivity was associated with loss to producers of $ 285 million and $ 240 million for consumers. Most of this $ 525 million industry loss was due to reduced milk production in test-positive herds.     

Counter current line absorption immunoelectrophoresis in an alternative diagnostic screening test to counter current immunoelectrophoresis in Aleutian disease (AD) eradication programs

Counter current immunoelectrophoresis (CCIE) is the diagnostic method used in the ongoing Aleutian disease virus eradication program on Danish mink farms. There has been an increasing demand for an alternative diagnostic test especially to evaluate suspected false positive CCIE reactions. We compared test results of a number of negative and positive mink sera in indirect counter current immunoelectrophoresis (ICCIE), counter current line absorption immunoelectrophoresis (CCLAIE) and radio immuno assay (RIA) with test results from counter current immunoelectrophoresis and found that counter current line absorption immunoelectrophoresis is the best alternative diagnostic screening test to counter current immunoelectrophoresis for Aleutian disease eradication programs. Not only proved the CCLAIE test to be useful for evaluation of doubtfully positive CCIE reactions, but it was found to have a higher sensitivity than the CCIE test.     

番鸭小鹅瘟病胶乳凝集试验与病毒分离的比较研究

应用GPV胶乳凝集方法（LPA）和病毒分离（VI）检测人工感染病例9份、健康对照6份,临床疑似送检病鸭28份。其中人工感染样品检测LPA和VI符合率为93.3%（14/15）;临床疑似样品检测LPA和VI符合率为89.3%（25/28）;两者总符合率达90.7%（39/43）。LPA方法具有简便（肉眼可判定,不需仪器）、快速（检测抗原20min内）、特异、准确等优点,比VI更适合临床使用。     

脂质体介导抗猪瘟病毒基因转染小鼠胚胎成纤维细胞的研究

本研究旨在探索脂质体介导抗猪瘟病毒基因重组质粒PGPUG/GFP/Neo转染小鼠胚胎成纤维细胞的条件,为构建疾病模型动物及抗猪瘟病毒转基因猪提供技术平台。本研究比较了不同的小鼠胚胎成纤维细胞分离培养方法,将培养至3~5代的胚胎儿成纤维细胞,通过脂质体(LiPofectamine^TM 2000)介导转染抗猪瘟病毒基因重组质粒PGPUG/GFP/Neo。结果显示,在所选用的不同浓度脂质体和重组质粒,以及不同转染时间的组合中,2 μL脂质体介导1.5 μg重组质粒,转染6 h时可获得19%的转染效率,极显著高于其他剂量和时间组合(P＜0.01)。这些结果表明,脂质体、重组质粒及转染时间的优化组合,能有效介导抗猪瘟病毒基因重组质粒PGPUG/GFP/Neo转染小鼠胚胎成纤维细胞。     

30个紫花苜蓿品种对苜蓿花叶病毒病的田间抗性初步研究

为了比较不同紫花苜蓿品种对病毒病的抗性,对30个紫花苜蓿品种的病毒病发病情况进行了田间调查,利用小RNA深度测序技术结合RT-PCR技术检测鉴定各紫花苜蓿品种中的病毒种类,并测定了各紫花苜蓿品种的发病率、病情指数、相对株高、相对单株干重、相对茎秆直径、相对叶面积、相对叶长和相对叶宽,综合比较各紫花苜蓿品种的抗性,并对部分指标进行系统聚类分析。结果表明:30个紫花苜蓿品种均只受到苜蓿花叶病毒(alfalfa mosaic virus, AMV)的侵染,所有紫花苜蓿品种的田间发病率均超过94%,病情指数范围为31.53～55.50;30个紫花苜蓿品种中,抗性较强的品种有1个,为射手2号,抗性一般的品种有6个,其余23个品种的抗性较差,分别占供试品种的3.33%,20.00%和76.67%;聚类分析结果表明,抗性较强的射手2号与其他紫花苜蓿品种的亲缘关系较远,与其杂交有可能产生较大的杂种优势。     

一株无血凝性兔出血症病毒的分离鉴定及其衣壳蛋白VP60基因的克隆和序列分析

从临床病料中分离鉴定出1株无血凝性的致病性兔出血症病毒（RHDV），命名为Yaan-1。HA试验测得该毒株原代及经兔体传3代后的肝毒在25℃、37℃和4℃时均无血凝性；但在琼脂扩散试验和对流免疫电泳中均可见分离株与常规RHDV高免血清形成清晰的沉淀线；电镜观察可见30nm左右的病毒粒子。采用RT-PCR方法将Yaan-1株的衣壳蛋白VP60基因进行了克隆测序，序列分析表明VP60基因序列全长1740bp，编码579个氨基酸，测序结果提交GenBank（注册号为DQ069280），并与世界各国的常规RHDV分离株进行比较，结果表明核苷酸同源性为90．0％～98．0％，氨基酸同源性为94．1％～99．0％，氨基酸变异多发生在衣壳蛋白C、E区；同时对Yaan-1株的分子量、等电点、疏水性、跨膜区等分子结构特征进行了分析。本研究首次报道了无血凝性RHDV中国分离株，丰富了地方毒株资源，为明确我国地方株的分子流行病学，以及对VP60蛋白分子特征研究提供了新材料。     

Isolation and biological properties of virulent subpopulations from lentogenic Newcastle disease virus strains

From each of two lentogenic Newcastle disease virus (NDV) strains of the type LaSota and Hitchner B1 a virulent subpopulation could be obtained. The two subpopulations were—in comparison to the two parent viruses—more resistant to the lipid solvent chloroform and more stable against thermal degradation. Also, the glycoproteins haemagglutinin and F (fusion) were more stable against thermal inactivation. Electron microscopic observations revealed in terms of size and morphology all of the characteristics of NDV. Both subpopulations possessed, however, the same elution kinetics as their respective parent strains. The intracerebral and intravenous pathogenicity indices as well as the mean death times of the two subpopulations allow to classify these viruses as virulent Newcastle disease viruses.