Pelargonium flower-break and pelargonium line pattern viruses in the Netherlands; purification,antiserum preparation,serological identification,and detection in pelargonium by ELISA

Two viruses, detected frequently in the Netherlands in pelargonium, were identified by serology and test plant reactions. Antisera were prepared and an ELISA procedure was developed to detect the viruses in pelargonium.One of the viruses, PFBV-N, proved to be pelargonium flower-break virus. With the antiserum to PFBV-N, it could be detected reliably throughout the year inPelargonium zonale Springtime Irene.The other virus, PLPV-N, was serologically closely related to pelargonium line pattern virus (PLPV) and to pelargonium ring pattern virus (PRPV), as were an old virus isolate from Saturnus, collected in the Netherlands in 1971 (L128), and PLPV isolates from Yugoslavia (PLPV-Y) and Denmark (PLPV-D). There were only minor differences in host-plant reactions between the virus isolates. Based on these tests, PLPV and PRPV are considered as isolates of the same virus, for which, for practical reasons, the name pelargonium line pattern virus is proposed.PLPV could be reliably detected by ELISA inP. zonale Springtime Irene and Amanda throughout the year with only a few exceptions. InPelargonium peltatum Tavira, however, reslts were erratic due to uneven distribution of virus in the plant. Best results were obtained when petioles of fully expanded leaves were tested.     

温室番茄无土多层栽培技术开发研究

本研究的目的在于验证由山东农业大学园艺系研制的“鲁SC-1型”无土栽培装置及其配套栽培技术在大面积温室番茄无土多层栽培的有效性与可行性。番茄亩产量达12616.2公斤,亩产值超出土壤栽培184.98%,虽然生产成本比土壤栽培明显增大达138.34%。但由于增产基数超出土壤栽培较大,所以,亩净产值和盈利值分别比土壤栽培提高156.82%和227.73%。成本占用盈利率以及劳动盈利率等也均有明显超出。     

Detection of squash mosaic virus in seeds of melon (Cucumis melo) by enzyme-linked immunosorbent assay (ELISA)

Squash mosaic virus (SqMV, comovirus) is seed-transmitted in severalCucurbitaceae. Therefore, the use of virus-free seed is important to prevent establishment of this virus in the Netherlands and to avoid spread to other countries.This study was undertaken to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of SqMV in melon seeds. An antiserum was produced to a serotype 1 isolate from melon. Two ELISA variants were investigated viz. an ELISA variant with simultaneous incubation of sample and enzyme conjugate (ELISA 1) and an ELISA variant with successive incubation of sample and enzyme conjugate (ELISA 2). The sensitivity of ELISA was tested by mixing fluor of ground infected and non-infected seeds in different proportions. SqMV was detected by both ELISA variants at dilutions of 1 160 (1 part of infected flour mixed with 159 parts of non-infected flour) or higher after a substrate incubation period of 4 h. However, ELISA 1 gave relatively higher absorbance values than ELISA 2 for nearly all dilutions. Since ELISA 1 is also faster than ELISA 2, ELISA 1 is advised for routine testing. In these test, using subsamples of 100 melon seeds SqMV is detected reliably. ELISA 1 is now used in the Netherlands for routine-indexing of melon seed lots for SqMV.Samenvatting Het pompoenemozaïekvirus gaat over met het zaad van verscheideneCucurbitaceae. Het gebruik van virusvrij zaad is belangrijk om te voorkomen dat het virus zijn intrede doet in Nederland en zich naar andere landen verspreidt.Een antiserum werd geproduceerd tegen een serotype 1 isolaat van meloen. Met behulp van dit antiserum werd een ELISA ontwikkeld om pompoenemozaïekvirus in zaden van meloen aan te tonen. Twee varianten van ELISA werden vergeleken, namelijk een variant waarbij monster en enzymconjugaat gelijktijdig geïncubeerd werden (ELISA 1) en een variant waarbij monster en enzymconjugaat na elkaar geïncubeerd werden (ELISA 2). De gevoeligheid van de ELISA varianten werd uitgetoetst door meel van zieke zaden in verschillende verhoudingen te mengen met meel van gezonde zaden. Het pompoenemozaïekvirus werd met beide ELISA varianten aangetoond in verdunningen van 1 160 (1 deel meel van zieke zaden gemengd met 159 delen meel van gezonde zaden) of hoger na 4 uur incubatie met substraat. ELISA 1 gaf doorgaans hogere extinctiewaarden dan ELISA 2 voor bijna alle verdunningen. Omdat ELISA 1 ook nog sneller is dan ELISA 2, wordt ELISA 1 aanbevolen voor routinematig gebruik. Wanneer voor routinematig gebruik 100 meloenezaden per submonster getoetst worden, kan het pompoenemozaïek virus betrouwbaar worden aangetoond. In Nederland worden momenteel per zaadpartij 20 submonsters van 100 zaden getoetst.     

A seroepidemiological study on bluetongue virus in dairy cattle in the central valley of California

A seroepidemiological study on bluetongue virus (BTV) infection in California dairy cattle was conducted to estimate the prevalence and distribution by age and season of BTV group-reactive antibodies and to look for possible associations between the presence of antibodies and cattle age or breed and farm. Between December 1985 and March 1987, a sample of cattle was tested at approximately two-month intervals for BTV group-reactive antibodies using an enzyme-linked immunosorbent assay (ELISA). Data taken during the month of December 1986 were used to evaluate possible associations between a positive antibody test and certain intrinsic (age, breed) and extrinsic (farm) factors.Univariate and multivariate statistical analyses using the -square test for associations and multiple logistic regression, respectively, were carried out for possible associations between positive antibody tests to BTV and each factor of interest. The strengths of the associations were determined using estimates of the odds ratio.Of the 3774 serum samples tested, 238 (6.3%) were from calves, 1045 (27.6%) were from heifers and 2492 (66.0%) were from cows. Seroprevalence varied from nil in calves on two occasions to over 90% on several occasions in cows. Cows consistently had higher prevalence rates than heifers or calves across all test dates (p<0.05). The seroprevalence of BTV group-reactive antibodies also showed a seasonal fluctuation, with the highest rates occurring during the warmer months of the year. These highest prevalence rates coincided with heavy activity of the known vector of BTV, Culicoides spp. Breed and farm effects were not statistically significant (p>0.05). With the exception of one farm, all cattle were of the Holstein breed, which reduced confidence in assessing any breed effect in this study. Relative estimates of the sensitivity and specificity of BTV ELISA were 87% and 100% respectively, compared to the standard agar gel immunodiffusion (AGID) test.The observations support previous findings of seasonal distribution of BTV antibodies and suggest an age relationship, whereby older cattle are more likely to be positive to BTV group-reactive antibodies than younger cattle.     

Age-related resistance to Pseudomonas syringae pv. tomato is associated with the transition to flowering in Arabidopsis and is effective against Peronospora parasitica

As plants mature it has been observed that some become more resistant to normally virulent pathogens. The ability to manifest the Age-Related Resistance (ARR) response in Arabidopsis to Pseudomonas syringae pathovars tomato (Pst) coincided with the transition to flowering in plants both delayed and accelerated in the transition to flowering. ARR was also associated with a change in PR-1 gene expression, such that young plants expressed PR-1 abundantly at 3 days post inoculation (dpi) while mature plants expressed much less. The Arabidopsis ARR response requires SA accumulation via isochorismate synthase (ICS1) [24]. ICS1 was expressed one dpi with virulent and avirulent Pst in both young and mature plants. The ARR response was also effective versus avirulent Pst providing an additional 4-fold limitation in bacterial growth. Arabidopsis ARR was found to be ineffective against two necrotrophs, Erwinia carotovora subspecies carotovora (bacterium) and Botrytis cinerea (fungus) and one obligate biotroph, Erysiphe cichoracearum (fungus). However, mature wild type, SA-deficient sid2 and NahG plants supported little growth of the obligate biotrophic oomycete, Peronospora parasitica. Therefore ARR to P. parasitica appears to be SA-independent, however the level of ARR resistance was somewhat reduced in these mutants in some experiments. Thus, there may be numerous defence pathways that contribute to adult plant resistance in Arabidopsis.     

柑橘抗CTV转基因与分子标记研究进展

综述了柑橘抗衰退病基因工程中两方面的研究进展。介绍多种来源于柑橘衰退病毒(Citrustriztezavirus,CTV)核酸序列的转基因柑橘和抗性种质资源中抗性基因的分子标记,以及所涉及的方法和遇到的问题。目前研究表明,虽然已成功实现对病毒衣壳蛋白(CP)等基因的转化和Ctv等抗性基因的标记,但尚未获得对CTV有高度抗性的转基因柑橘,而抗性基因亦不能实现定点克隆和转化。因此上述两方面研究还有待深入。     

H5N1亚型禽流感病毒NS1基因的原核表达及其ELISA检测方法的建立

采用RT-PCR技术扩增了禽流感病毒（AIV）A／Goose／HLJ／p46／2003（H5N1）的NSl基因，将其克隆于融合蛋白表达载体pMAL-c2X上，转化DH5α大肠埃希氏菌感受态细胞，经BamHⅠ和HindⅢ双酶切鉴定及序列分析，表明筛选到了重组质粒pc2X-NS1。SDS-PAGE电泳结果显示，重组质粒转化TB1大肠埃希氏菌后，经0．3mmol／L的IPTG诱导，融合蛋白MBP-NS1得到大量表达，融合蛋白以可溶形式存在，分子质量约为67ku。Western-blotting检测结果表明，融合蛋白MBP-NS1能够与H5N1亚型AIV活病毒感染康复鸭血清发生特异性反应，而不能够与H5N1亚型AIV灭活疫苗免疫鸭血清发生反应。试验初步建立了以纯化的融合蛋白MBP-NS1为包被抗原的间接ELISA检测方法，为AIV灭活疫苗免疫家禽与AIV感染家禽的鉴别诊断奠定了基础。     

猪血凝性脑脊髓炎病毒抗体的调查

应用血凝和血凝抑制试验检测了从吉林省部分地区采集的猪血清中血凝性脑脊髓炎病毒（HEV）抗体。结果，212份样品中有94份呈现HEV抗体阳性反应，阳性率高达44．3％。被采集血清的猪未表现临床症状，说明该地区的猪群中存在HEV隐性感染。     

猪水泡病病毒结构蛋白基因的克隆及其蛋白二级结构和淋巴细胞表位的预测

以免疫信息学和反向疫苗学为理论根据，利用RT-PCR法获得了猪水泡病病毒（SVDV）HK／70株的基因组序列并测序，应用生物信息学相关软件和方法，对SVDV结构蛋白的二级结构的不同方面及其抗原特异性淋巴细胞表位进行了预测，综合评价了SVDV结构蛋白的抗原特异性细胞表位，并计算出一些潜在的抗原位点。结果表明，VP1蛋白含有的细胞优势抗原表位最多，但其他结构蛋白也含有抗原特异性淋巴细胞表位，有的甚至有可能成为优势抗原表位或对优势抗原表位有协同作用。     

鸡传染性腔上囊病病毒VP2基因在昆虫细胞中的表达

为了深入研究鸡传染性腔上囊病病毒（IBDV）VP2基因的结构和功能,利用Bac-to-Bac系统研制出含有VP2基因的重组杆状病毒rBac-VP2,将rBac-VP2感染Sf9细胞,并用抗IBDV VP2特异性单克隆抗体经间接免疫荧光试验检测,证实感染重组病毒Sf9细胞能高效表达IBDV VP2基因产物;Western-blotting分析结果表明,VP2基因表达产物的分子质量约为40 ku.