一个马铃薯Y病毒山东分离物的分离与鉴定

 从具有典型花叶症状的马铃薯叶片中分离到马铃薯Y病毒(Potato virus Y,PVY)(本文称PVY-SD-TA分离物),扩繁后,提纯病毒,电镜下可观察到700~900 nm×11 nm的病毒粒体,病组织超薄切片观察可见风轮状的内含体结构,寄主反应特性研究表明其能侵染2科13种植物。SDS-PAGE电泳检测病毒编码的外壳蛋白亚基的分子量为33 kDa。以PVY-SD-TA基因组RNA为模板,应用RT-PCR方法和特异引物合成了外壳蛋白基因。对cDNA全序列分析表明,PVY-SD-TA CP基因核苷酸序列与N株系的同源性为96%,与GenBank中登录序列号为AJ390306的O株系分离物的同源性最高,为99%;与国内不同学者报道的PVY中国流行株的同源性分别为96%,97%和98%。通过以上生物学特性和分子水平的研究将PVY-SD-TA鉴定为普通株系(PVY^O株系)。     

利用RT-PCR检测库尔勒香梨苹果茎痘病毒的研究

 以库尔勒香梨新鲜、冷藏、冷冻叶片和皮层为材料,对提取双链RNA (dsRNA)的2种方法和提取总RNA的3种方法进行了分析比较,并对总RNA的提取方法进行了改进,获得了纯度较高、完整性较好的dsRNA和总RNA,在此基础上进行了反转录(RT)和PCR扩增。在国内首次完成了对苹果茎痘病毒(ASPV)的RT-PCR检测,建立了ASPV有效RT-PCR反应体系。用此体系扩增到ASPV一个长约316 bp的片段。实验表明以dsRNA和总RNA为模板均能成功进行RT-PCR检测,且dsRNA优于总RNA。     

Non-structural plum pox potyvirus proteins detected by immunogold labelling

Plum pox potyvirus (PPV) induces in infected Nicotiana clevelandii cells characteristic crystalline inclusions known as nuclear inclusions (NI) when located in the nucleus and as dense material (Dm) when located in the cytoplasm. Crystalline inclusions contain protease (NIa) and RNA-dependent RNA polymerase (NIb) proteins. It is now well established for all potyviruses that cylindrical inclusions contain CI helicase ATPase protein (Martin et al., 1992). The intracellular location of other non-structural PPV proteins remains unknown. Using Escherichia coli expression vectors, specific antibodies were obtained against P1, P3, 6K2 and NIb PPV proteins for which antibodies were not yet available. As expected, NIb antiserum labelled crystalline inclusions. P1, P3 and 6K2 proteins were present in both types of crystalline inclusions found in the nucleus and in the cytoplasm of PPV-infected leaves of N. clevelandii, suggesting that nuclear inclusions and dense material were composed of the same proteins. This composition is discussed.     

Phylogenetic relationships between rice dwarf phytoreovirus isolates from five countries

Rice dwarf virus isolates were collected from several locations in Japan, the Philippines, China, Nepal and Korea. Genomic dsRNA segment profiles in polyacrylamide gel electrophoresis differed among the isolates. There were less differences in the profiles between isolates from Japan and Korea than in those between these two Countries and others. Nucleic acid hybridization was used to examine the extent of genomic variation. Full-length cDNAs to all genomic segments encoding non-structural proteins (S4, S6, S9, S10, S11 and S12) were synthesized from two Japanese isolates, and were used for dot-blot hybridization. Hybridizations using probes generated from the full-length cDNA clones failed to differentiate isolates from different geographical areas. However, cDNA probes covering a variable region of S12 were able to distinguish Japanese and Korean isolates from those of other countries. Phylogenetic tree analysis based on the amino acid sequence of P12 encoded by S12 grouped Japanese and Korean isolates together. The Chinese isolates from two different locations (Yunnan and Fujian) were closely related to each other, and were the most distantly related to Japanese and Korean isolates.     

Biological and molecular characterization of Tomato spotted wilt Virus in Israel

Received April 24, 1997; received in final form June 29, 1997. Symptoms resembling tomato spotted wilt virus (TSWV) infections were documented among ornamental and vegetable crops in commercial greenhouses and open fields in Israel. Plants exhibiting these symptoms were collected from January 1992 to December 1996. Among cultivated plants analyzed for TSWV by enzyme-linked immunosorbent assay (ELISA), 19 species representing five families were found to be infected; natural infection was also recorded in six plant species of weeds. Virus identity was characterized by host range, serology and electron microscopy. Serological reaction with the isolates, found in Israel, using antisera from different sources as well as the sequence analysis of the nucleocapsid gene, demonstrated that the Israeli isolates of TSWV are a member of tospovirus serogroup I, type I (BR-01 strain). No virus transmission was found in seeds collected from virus-infected vegetable and ornamental crops. A non-radioactive molecular probe derived from the cloned nucleocapsid isolate enables specific detection of the virus in crude sap from infected plants. The detection of TSWV in Israel constitutes a severe potential threat to the ornamental and vegetable industry.     

狂犬病毒中和抗体检测试验中病毒回归试验的重要性

狂犬病毒中和试验的病毒回归试验表明,病毒攻毒液的实际剂量与理论剂量不完全一致.建议在新版<中华人民共和国兽用生物制品质量标准>中增加对病毒攻毒实际剂量范围的规定,以使结果的判定更为合理.     

检测猪流行性腹泻病毒的R-PCR方法的建立

根据猪流行性腹泻病毒 (PEDV)的N基因自行设计和合成了一对可扩增长度为 641bp目的片段的引物 ,成功地建立了检测的猪流行性腹泻病毒的RT PCR方法。对猪轮状病毒 (PRV)、猪传染性胃肠炎病毒 (TGEV)的RT PCR检测结果均呈阴性。对PEDV JS株的RT PCR产物的序列分析表明 ,与CV777株的同源性为 97 3 %。     

圆点印迹法检测葡萄斑点病毒技术研究

采用圆点印迹法检测GFkV，结果表明：Tris-HCl和PBS在提取GFkV时效果相同，在带毒葡萄植株中韧皮部的GFkV含量较高。     

Factors affecting the development of disease symptoms in potatoes infected by <Emphasis Type="Italic">Tobacco rattle virus</Emphasis>

Infection of potato plants with Tobacco rattle virus by its nematode vector can have different outcomes, including the development of spraing symptoms in progeny tubers. A novel syndrome described here comprises a generalized mottle in leaves on all stems, together with virtually total failure to produce daughter tubers. The outcome of infection depends partly on the potato genotype, but field trials with 15 varieties showed that the incidence and severity of spraing symptoms at three sites differed. There was no evidence that this was due to differences in virulence among the virus isolates at the sites, but it was probably the result of environmental differences that influenced the numbers and activity of the vector nematodes. At the most severely affected site, spraing symptoms were found in all varieties tested, except Record, including several that were not affected at the other two sites. Taking both severity and incidence of symptoms into account, the ranking of varieties was similar at each site. The incidence of spraing symptoms was greater in larger than in smaller tubers, and increased with later harvest dates, but did not increase when early harvested tubers were stored. Tobacco rattle viruswas detected in the roots of many of the weeds growing at one of the sites and in the roots of a few plants of the subsequent barley crop.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.