烟草环斑病毒的定量检测技术研究

目的:建立特异、灵敏、快速的TaqMam实时荧光定量PCR方法,用于烟草环斑病毒(TRSV)的定量检测。方法:用纳米磁珠法提取病毒RNA,构建包含烟草环斑病毒全CP序列的质粒标准品。根据CP保守序列设计特异性的引物和TaqMam荧光探针,构建标准曲线,建立TRSV的实时荧光绝对定量PCR方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:建立的方法特异性好,与南芥菜花叶病毒、马铃薯X病毒和马铃薯Y病毒均无交叉反应;至少能检测到767个病毒拷贝,灵敏度比普通PCR高100倍;同一样品试验内及试验间重复性实验的变异系数均小于3%,重复性好;检测结果准确可靠,构建的标准曲线有较好的线性关系(R2=0.997)。结论:建立的TRSV TaqMan实时荧光定量PCR检测方法可满足口岸高通量、快速、准确的检验检疫要求。     

病毒诱导的PVX cp转基因沉默及其DNA甲基化

利用PCR方法获得了马铃薯X病毒(PVX)外壳蛋白(CP)基因(cp),并将其构建到植物表达载体中,利用农杆菌介导的叶盘法转化烟草(Nicotiana tabacum L.)。Northern杂交及Run on实验表明有3株转基因烟草发生了转录后基因沉默。发生沉默的cp基因的甲基化分析结果表明,发生转录后基因沉默的cp基因发生了不同程度的甲基化,说明DNA甲基化并没有完全抑制cp基因的转录。利用PVX病毒对外壳蛋白正常表达的转基因烟草进行接毒,Northern杂交检测结果表明,病毒诱导cp发生了基因沉默。进一步的Run on结果表明,转基因烟草中cp基因在沉默前后转录速率并没有发生变化,说明病毒诱导的沉默是一种转录后沉默。对cp基因沉默前后的甲基化分析表明,病毒的侵染导致了cp基因甲基化程度的增加。     

病毒诱导的PVX cp转基因沉默及其DNA甲基化

利用PCR方法获得了马铃薯X病毒(PVX)外壳蛋白(CP)基因(cp),并将其构建到植物表达载体中,利用农杆菌介导的叶盘法转化烟草(Nicotiana tabacum L.).Northern杂交及Run on实验表明有3株转基因烟草发生了转录后基因沉默.发生沉默的cp基因的甲基化分析结果表明,发生转录后基因沉默的cp基因发生了不同程度的甲基化,说明DNA甲基化并没有完全抑制cp基因的转录.利用PVX病毒对外壳蛋白正常表达的转基因烟草进行接毒,Northern杂交检测结果表明,病毒诱导cp发生了基因沉默.进一步的Run on结果表明,转基因烟草中cp基因在沉默前后转录速率并没有发生变化,说明病毒诱导的沉默是一种转录后沉默.对cp基因沉默前后的甲基化分析表明,病毒的侵染导致了cp基因甲基化程度的增加.     

抗黄瓜花叶病毒单克隆抗体细胞株的建立及其抗体鉴定

用黄瓜花叶病毒(CMV)免疫的BALB／C小鼠脾细胞与骨髓瘤细胞SP2／0-Ag-14,经5次细胞融合,6次有限稀释,获得1株6个稳定分泌cMV单克隆抗体的杂交瘤细胞培养物,所分泌的单克隆抗体均属IgM亚类。试验结果证明,6个单克隆抗体对cMV是特异的,对CMV-Q、CMV—P和cMv—B呈阳性反应,对cMV一6和番茄不孕病毒(TAV)呈阴性反应,对所用的其他8种植物病毒：南瓜花叶病毒(0qMV)、绒毛烟斑驳病毒(VT№V)、大毒条纹花叶病毒(BsMv)、马铃薯病毒x(PVx)、马铃薯病毒Y(PVY)、玉米矮箍花叶病毒(MDMV)、大豆花叶 病毒(sMV)和烟草花叶病毒(TMV)均无交叉反应。     

马铃薯Y病毒蚜传辅助成分介导PVX/PVY协生作用

构建了马铃薯Y病毒中国株系（PVY－C）蚜传辅助成分（HC－Pro）基因的正义、反义和缺失三种植物表达载体,通过农杆菌介导法转化烟草品种NC89。Southern blot分析表明,HC－Pro基因及其突变体已经整合到烟草染色体中,Western blot分析证明,正义HC－Pro基因及其缺失突变体在转基因烟草中有表达产物,攻毒试验结果表明,转正义,HC－Pro基因及其缺失突变体不仅能够提高T1转基因烟草中PVY－C的病毒积累和致病,而且对异源病毒PVX具有同样的作用,而转反义HC－Pro基因烟草对PVY－C和PVX的致病性无影响,因此,PVY－C HC－Pro基因介导PVX／PVY的协作作用。     

葡萄试管苗光自养培养快速繁殖

葡萄是世界上栽培面积和总产量都占第一位的果树。有关葡萄茎尖培养、侧芽培养和无 病毒苗的试管繁殖都有报道。但是试管苗移栽成活率一般较低而且很不稳定,这就使葡萄试管苗(特别是脱病毒试管苗)快速繁殖在生产上的应用受到很大限制。本试验利用光自养培养获得的葡萄试管苗生长发育正常,移栽前练苗时培养基不易污染,可适当延长练苗时间,并且,此试管苗对移栽后的环境条件具有很强的适应能力,成活率高,生长速度快。我们     

马铃薯Y病毒HC-Pro中心区域在病毒协生作用中的主导地位

利用PCR方法获得了马铃薯病毒中国株系（PVY－C）HC－Pro基因的5个缺失突变体,构建了相应的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens)介导法转化了烟草品种K326(Nicotina tabacum cv.k326)。PCR和Southern blot分析证明了HC－Pro基因及其缺失突变体已整合到烟草基因组中,Western blot表明它们在转基因烟草中得到了表达。侵染性试验发现HC－Pro中心区域介导转基因烟草中PVC－C和黄瓜花叶病毒（CMV）、PVY－C和马铃薯X病毒（PVX）之间的协生作用,从而明确了PVY－C HC－Pro中心区域为病毒协生作用的功能区域。     

马铃薯Y病毒HC-Pro中心区域在病毒协生作用中的主导地位

利用PCR方法获得了马铃薯病毒中国株系(PVY-C)HC-Pro基因的5个缺失突变体,构建了相应的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens)介导法转化了烟草品种K₃₂₆(Nicotina tabacum cv.K₃₂₆)。PCR和Southern blot分析证明了HCPro基因及其缺失突变体已整合到烟草基因组中,Western blot表明它们在转基因烟草中得到了表达。侵染性试验发现HCPro中心区域介导转基因烟草中PVY-C和黄瓜花叶病毒(CMV)、PVYC和马铃薯X病毒(PVX)之间的协生作用,从而明确了PVY-C HC-Pro中心区域为病毒协生作用的功能区域。     

酶联板重复使用的初步研究

本实验用10种溶液处理仙人指×病毒(CVX)、齿兰环斑病毒(ORSV)、烟草花叶病毒(TMV)、烟草脆裂病毒(TRV)进行间接ELISA试验后的微板,其中以SDS处理效果较好,微板可重复使用1—3次,并基本保持原板检测病毒的灵敏度,无假阳性反应出现,底色上升也较缓慢。     

重庆烟草地区ELISA检测

为调查重庆主要烟草地区的病毒病种类,采用没脸免疫吸附法(ELISA)对采自重庆涪陵、彭水、万州、黔江、武隆、酉阳的258份的烟草样品进行了病毒病种类的检测.检测结果表明,其中274份样品呈阳性。主要受到7种病毒病的侵染,主要包括:烟草花叶病毒(Tobacco mosaic virus,TMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、马铃薯Y病毒(Potato virus Y,PVY)、马铃薯X病毒(Potato virus X,PVX)、芜菁花叶病毒(Turnip mosaic virus,TuMV)、蚕豆萎蔫病毒2号Broad bean wilt virus 2,BBWV-2)、烟草蚀纹病毒(Tobacco etch virus,TEV)。其中TMV的检出率最高,达50%,已成为重庆地区烟草优势病原。     

共生因子PABA对P.gingivalis生长代谢影响的实验培养基的筛选

目的 为了研究其生因子PABA在龈下菌斑微生态平衡中的作用,拟筛选出一种既能满足P.gingivalis基本生长要求,又含最低浓度PABA的培养基。方法 在Carlsson培养基中加入营养成分进行改良,将BHI培养基系列稀释,共6个培养基系列进行筛选。结果 P.ginsivalis在改良Carlsson培养基中不生长,在BHI系列稀释培养基中只有1／2浓度才达到理想效果。结论 将稀释至原油质1／2的BHI培养基作为本研究的实验培养基。     

Virion proteomics of large DNA viruses

Large DNA viruses normally have complex structures with many of protein components derived from both viral and host origins. The development in proteomics, especially mass spectrometry identification techniques provide powerful tools for analyzing large viruses. In this review, we have summarized the recent achievements on proteomic studies of large DNA viruses, such as herpesvirus, poxvirus, nimavirus and baculoviruse. The proteomics of baculovirus occlusion-derived virions (ODV) were emphasized. Different mass spectrometry techniques used on ,carious baculoviruses were introduced, and the identified structurally associated proteins of baculoviruses are summarized.     

Lifestyles of plant viruses

The vast majority of well-characterized eukaryotic viruses are those that cause acute or chronic infections in humans and domestic plants and animals. However, asymptomatic persistent viruses have been described in animals, and are thought to be sources for emerging acute viruses. Although not previously described in these terms, there are also many viruses of plants that maintain a persistent lifestyle. They have been largely ignored because they do not generally cause disease. The persistent viruses in plants belong to the family Partitiviridae or the genus Endornavirus. These groups also have members that infect fungi. Phylogenetic analysis of the partitivirus RNA-dependent RNA polymerase genes suggests that these viruses have been transmitted between plants and fungi. Additional families of viruses traditionally thought to be fungal viruses are also found frequently in plants, and may represent a similar scenario of persistent lifestyles, and some acute or chronic viruses of crop plants may maintain a persistent lifestyle in wild plants. Persistent, chronic and acute lifestyles of plant viruses are contrasted from both a functional and evolutionary perspective, and the potential role of these lifestyles in host evolution is discussed.     

Plasminogen-binding activity of neuraminidase determines the pathogenicity of influenza A virus

When expressed in vitro, the neuraminidase (NA) of A/WSN/33 (WSN) virus binds and sequesters plasminogen on the cell surface, leading to enhanced cleavage of the viral hemagglutinin. To obtain direct evidence that the plasminogen-binding activity of the NA enhances the pathogenicity of WSN virus, we generated mutant viruses whose NAs lacked plasminogen-binding activity because of a mutation at the C terminus, from Lys to Arg or Leu. In the presence of trypsin, these mutant viruses replicated similarly to wild-type virus in cell culture. By contrast, in the presence of plasminogen, the mutant viruses failed to undergo multiple cycles of replication while the wild-type virus grew normally. The mutant viruses showed attenuated growth in mice and failed to grow at all in the brain. Furthermore, another mutant WSN virus, possessing an NA with a glycosylation site at position 130 (146 in N2 numbering), leading to the loss of neurovirulence, failed to grow in cell culture in the presence of plasminogen. We conclude that the plasminogen-binding activity of the WSN NA determines its pathogenicity in mice.     

CHARACTERIZATION OF HaRNAV,A SINGLE‐STRANDED RNA VIRUS CAUSING LYSIS OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)1

HaRNAV, a novel virus that infects the toxic bloom‐forming alga Heterosigma akashiwo (Hada) Hada ex Hada et Chihara, was characterized based on morphology, pathology, nucleic acid type, structural proteins, and the range of host strains that it infects. HaRNAV is a 25‐nm single‐stranded RNA (ssRNA) virus with a genome size of approximately 9100 nucleotides. This is the first report of an ssRNA virus that causes lysis of a phytoplankton species. The virus particle is sensitive to chloroform and contains at least five structural proteins ranging in apparent size from 24 to 34 kDa. HaRNAV infection causes swelling of the endoplasmic reticulum and progeny virus particles assemble in the cytoplasm of the host, frequently in crystalline arrays. The infectivity of HaRNAV was tested against 15 strains of H. akashiwo isolated from Japanese waters, the Northeast Pacific, and the Northwest Atlantic. HaRNAV caused lysis of three strains from the Northeast Pacific and two strains from Japan but none from the Northwest Atlantic. The characterization of HaRNAV demonstrates that HaRNAV is a novel type of phytoplankton virus but has some similarities with plant viruses belonging to the Sequiviridae and to other known ssRNA viruses. Further genomic analysis, however, is necessary to determine any phylogenetic relationships. The discovery of HaRNAV emphasizes the diversity of H. akashiwo viral pathogens and, more importantly, algal–virus pathogens and the complexity of virus–host interactions in the environment.     

Comparison of bacteriophage and enteric virus removal in pilot scale activated sludge plants

AIMS: The aim of this experimental study was to determine comparatively the removal of two types of bacteriophages, a somatic coliphage and an F-specific RNA phage and of three types of enteric viruses, hepatitis A virus (HAV), poliovirus and rotavirus during sewage treatment by activated sludge using laboratory pilot plants. METHODS AND RESULTS: The cultivable simian rotavirus SA11, the HAV HM 175/18f cytopathic strain and poliovirus were quantified by cell culture. The bacteriophages were quantified by plaque formation on the host bacterium in agar medium. In each experiment, two pilots simulating full-scale activated sludge plants were inoculated with viruses at known concentrations, and mixed liquor and effluent samples were analysed regularly. In the mixed liquor, liquid and solid fractions were analysed separately. The viral behaviour in both the liquid and solid phases was similar between pilots of each experiment. Viral concentrations decreased rapidly following viral injection in the pilots. Ten minutes after the injections, viral concentrations in the liquid phase had decreased from 1.0 +/- 0.4 log to 2.2 +/- 0.3 log. Poliovirus and HAV were predominantly adsorbed on the solid matters of the mixed liquor while rotavirus was not detectable in the solid phase. In our model, the estimated mean log viral reductions after 3-day experiment were 9.2 +/- 0.4 for rotavirus, 6.6 +/- 2.4 for poliovirus, 5.9 +/- 3.5 for HAV, 3.2 +/- 1.2 for MS2 and 2.3 +/- 0.5 for PhiX174. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the pilots are useful models to assess the removal of infectious enteric viruses and bacteriophages by activated sludge treatment. Our results show the efficacy of the activated sludge treatment on the five viruses and suggest that coliphages could be an acceptable indicator of viral removal in this treatment system.     

Technical advancement in plant virus diagnosis – an appraisal

Plants infected by a wide range of viruses often cause important agronomic and economic losses worldwide. Detection of plant viruses is becoming more challenging as globalisation of trade, particularly the ornamental plants, and the potential effects of climate change facilitate movement of viruses and their vectors, thereby transforming the diagnostic landscape. Under such circumstances, detection of viruses at premature stages of infection by use of rapid, sensitive and accurate detection methods seems crucial to ensure safe and sustainable agriculture and as such reduces economic losses. For proper identification, detection technique needs to be rapid, most accurate and inexpensive, as it forms the key step in developing appropriate practical solutions to manage plant virus diseases that pose continuous threats to the viability of certain horticultural and agricultural industries. This review is concerned with the advancement in techniques in the diagnosis of viruses in plants.     

EXPERIMENTAL EVOLUTION OF AN EMERGING PLANT VIRUS IN HOST GENOTYPES THAT DIFFER IN THEIR SUSCEPTIBILITY TO INFECTION

This study evaluates the extent to which genetic differences among host individuals from the same species condition the evolution of a plant RNA virus. We performed a threefold replicated evolution experiment in which Tobacco etch potyvirus isolate At17b (TEV‐At17b), adapted to Arabidopsis thaliana ecotype Ler‐0, was serially passaged in five genetically heterogeneous ecotypes of A. thaliana. After 15 passages we found that evolved viruses improved their fitness, showed higher infectivity and stronger virulence in their local host ecotypes. The genome of evolved lineages was sequenced and putative adaptive mutations identified. Host‐driven convergent mutations have been identified. Evidences supported selection for increased translational efficiency. Next, we sought for the specificity of virus adaptation by infecting all five ecotypes with all 15 evolved virus populations. We found that some ecotypes were more permissive to infection than others, and that some evolved virus isolates were more specialist/generalist than others. The bipartite network linking ecotypes with evolved viruses was significantly nested but not modular, suggesting that hard‐to‐infect ecotypes were infected by generalist viruses whereas easy‐to‐infect ecotypes were infected by all viruses, as predicted by a gene‐for‐gene model of infection.     

中国对虾中一种球状病毒的分离提纯与检测

从人工养殖生长比较缓慢的对虾与病虾的肝胰腺和消化道中,分离出一种大小为８０ｎｍ的球状病毒。同时采用超薄切片技术可在对虾中肠细胞中发现大量的球状病毒,这些病毒聚集成片,在同一细胞内的细胞器,如线粒体等均发生病变。此外,将分离提取的球状病毒进行感染试验,感染死亡率为６０％。并且从感染致死的对虾消化腺及肠道中,同样可以观察到大小相同、形态一致的球状病毒颗粒。