Detection and characterisation of bean yellow mosaic virus in corms of Gladiolus grandiflorus

Bean yellow mosaic virus (BYMV) could not be detected in corms of infected gladioli unless they were cut 6–60 days before testing. Detection after cutting was time- and temperature-dependent, was restricted to the cut area, and varied among cultivars. Virus could be recovered from uncut corms after storage for over 2 yr at 6 ^oC. BYMV in corms could be detected by enzyme-linked immunosorbent assay and by immunosorbent electron microscopy with antisera against a gladiolus isolate purified from gladiolus leaves or corms. It could not be detected in corms with antiserum against a lupin isolate which readily detected BYMV in gladiolus leaves. Protein subunits of corm-BYMV banded in SDS-PAGE as a single 31 000 dalton polypeptide, while leaf-BYMV produced a major 34 000 and several smaller polypeptides. Both major polypeptides retained the different serological properties of their source virions but their peptide maps indicated a common origin. It is suggested that the smaller polypeptide from corm-BYMV is a stable cleavage product of the intact leaf-BYMV coat subunits. Corm-BYMV, although lacking some of the antigenic properties of leaf-BYMV, was still infective.     

Detection of bean yellow mosaic virus in gladioli corms by the polymerase chain reaction

The polymerase chain reaction (PCR) method readily detected bean yellow mosaic virus (BYMV) in gladioli leaves, but in initial tests PCR did not detect virus in corm tissue. Extracts of RN A from corm tissue were shown to inhibit the amplification of viral sequences when added to a PCR reaction. An additional purification step for the RNA extracts using a Sephadex G-50 column eliminated the inhibitory effect and enabled PCR to amplify and detect viral RNA in corm tissue preparations.     

猪FGL2基因cDNA末端序列检测及结构分析

检测猪FGL2基因cDNA末端序列并对该基因结构初步分析。α-32P dCTP放射性同位素标记cDNA探针筛选猪基因组DNA文库;cDNA末端快速扩增(rapid amplification of cDNA end,RACE)。以猪正常小肠及心脏组织提取新鲜总RNA,反转录后作为模板,设计基因特异性引物,采用Advantage 2 聚合酶混合物进行PCR扩增;依据猪与人FGL2基因3′端已知同源序列设计PCR上游引物,以人FGL2基因3′末端序列设计下游引物,以猪基因组DNA为模板采用Advantage 2 聚合酶混合物进行PCR反应;PCR载体重组质粒DNA亚克隆扩增。同位素探针未能筛选到特异阳性克隆,RACE反应检测到特异性转录起始位置及第一个转录终止位置,但仍未检测到第二个转录终止位置。猪基因组DNA行PCR扩增成功检测到猪FGL2基因3′末端未知序列及第二个转录终止位置。     

Synthesis of encephalomyocarditis virus in a cell-free system: from DNA to RNA virus in one tube

Virus particles are used in vaccination, drug delivery, and material sciences. Here we devised a system where the RNA virus encephalomyocarditis virus (EMCV) is synthesized from DNA templates in vitro. When a plasmid or a PCR product harboring the full-length cDNA of EMCV in the T7 promoter/terminator unit was incubated in a HeLa cell extract supplemented with T7 RNA polymerase, EMCV was produced within 4 h at an efficiency of over 10-fold compared with the system programmed with the EMCV RNA. The EMCV RNA transcribed by the virally encoded RNA-dependent RNA polymerase was predominantly incorporated into the EMCV particle even in the presence of a larger amount of the EMCV RNA transcribed by T7 RNA polymerase from the plasmid.     

蛋白激酶抑制剂Flavopiridol对流感病毒复制的体外抑制作用

【目的】在细胞水平上研究黄酮类化合物flavopiridol的抗流感病毒效果,初步探索了其抗流感病毒的机制。【方法】首先用Western blot初步检测了在蛋白激酶抑制剂flavopiridol处理下流感病毒NP和M1蛋白的水平,然后通过免疫荧光实验观察了宿主细胞中流感病毒vRNP的合成,又利用噬斑实验检测了flavopiridol对病毒复制的影响,最后通过检测flavopiridol处理的宿主细胞内RNA聚合酶Ⅱ的磷酸化状态和病毒各种RNA的合成量,探究了flavopiridol抑制流感病毒复制的机理。【结果】结果表明,flavopiridol在细胞水平上可以显著抑制流感病毒蛋白质和vRNP的合成及病毒的复制,同时flavopiridol也可以抑制宿主RNA聚合酶Ⅱ大亚基CTD结构域七肽重复序列中的2位丝氨酸的磷酸化来抑制聚合酶的转录延伸活性,显著地减少病毒vRNA的合成。【结论】Flavopiridol可以通过抑制宿主细胞RNA聚合酶Ⅱ的转录延伸活性有效地抑制流感病毒的复制。     

Use of the Reverse Transcription-Polymerase Chain Reaction for the Detection of Pelargonium Flower Break Carmovirus

A polymerase chain reaction (PCR)-based assay was used to detect pelargonium flower break carmovirus (PFBV) in total RNA extractions made from infected Pelargonium plants. Extracts were reverse transcribed (RT) and the resultant cDNA was amplified by PCR, using oligonucleotide primers specific for 343, 510 and 832 base pair fragments of the RNA-dependent RNA polymerase gene of PFBV.
The specificity and sensitivity of RT-PCR were compared with the enzyme-linked immunosorbent assay (ELISA) for the detection of PFBV in Pelargonium tissues. The virus could be detected efficiently in high dilutions of sap from infected plants and at low concentrations of purified virus. Although ELISA is a powerful tool for virus detection, RT-PCR was over 1000 times more sensitive in detecting PFBV in leaf extracts of infected Pelargonium than was ELISA. The limit of detecting PFBV RNA by RT-PCR was 200 fg, compared with 200 pg of virus by ELISA.     

应用原位逆转录PCR技术检测石蜡切片中HCVRNA

利用原位逆转录聚合酶链式反应（ＩＳＲｔＰＣＲ）技术检测了１５例肝细胞癌及其癌旁肝组织中丙型肝炎病毒（ＨＣＶ）ＲＮＡ的定位及分布,并探讨影响实验结果的重要因素。结果表明ＨＣＶＲＮＡ阳性信号主要位于肝细胞和癌细胞胞浆,胞核几乎阴性。影响结果的主要因素包括所用蛋白酶Ｋ浓度,消化切片的时间,组织固定所用固定剂的选择及ＰＣＲ扩增的循环次数等     

Effects of cereal borders, admixture with cereals and plant density on the spread of bean yellow mosaic potyvirus into narrow-leafed lupins (Lupinus angustifolius)

In studies of virus control measures, field experiments in 1987–1991 investigated the effects of cereal and fallow borders, admixture with cereals and plant density on spread of bean yellow mosaic potyvirus (BYMV) from pastures dominated by subterranean clover (Trifolium subterraneum) into plots of narrow-leafed lupins (Lupinus angustifolius). Virus spread was mainly monocyclic because BYMV killed infected lupin plants and between systemic movement and death there was only a brief period for BYMV acquisition and transmission to other plants by vector aphids. In plots with cereal borders, the rate and extent of BYMV spread into the lupins was decreased; at final assessment the numbers of infected plants were 43–60% less than in plots with fallow borders. Admixture with cereals also decreased the rate and extent of BYMV spread into lupin plots, numbers of infected plants being decreased by 76–96% at the time of final assessment. When lupins were sown at different seeding rates to generate a range of plant densities and weeds were removed, high densities decreased BYMV infection. The higher incidences of BYMV infection in sparse stands were attributed partly to smaller plant numbers and partly to incoming viruliferous vector aphids being more attracted to plants with bare earth around them, than to a plant canopy. BYMV infection decreased grain yield of samples from infected lupin plants by 94–100%. In plots with 34% infection and sparse stands, grain yield was decreased by about one third. Plotted progress curves for the accumulated numbers of alate aphids of the BYMV vector species Acyrthosiphon kondoi and Myzus persicae resembled those for numbers of BYMV infected plants in 1990, but in 1991 only the curve plotted for M. persicae did so. There was a 2 week delay between the curves for aphid numbers and virus counts which reflected the time taken for obvious systemic necrotic symptoms to develop in lupins.