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.     

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.     

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。     

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.     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001     

A Cucumber mosaic virus Isolated from Silene armeria

A Cucumber mosaic virus was newly isolated from Silene armeria and was characterized by biological, serological and molecular biological methods. Received 4 July 2001/ Accepted in revised form 28 August 2001     

The Complete Nucleotide Sequence of a Japanese Isolate of White clover mosaic virus Strain RC

The complete nucleotide sequence was determined for genomic RNA of White clover mosaic virus (WClMV-RC) isolated from red clover (Trifolium pratense) in Japan, It is 5843 nucleotides in length, excluding the poly(A) tail at the 3' terminus. Similar to other potexviruses, it contains five open reading frames (ORFs 1 through 5), which putatively encode an RNA-dependent RNA polymerase (RdRp) (147 kDa), a triple gene block (TGB) (26 kDa/13 kDa/7 kDa), and a coat protein (CP) (22 kDa), respectively. The deduced amino acid sequence of the WClMV-RC CP was identical to that of WClMV-O, one of two New Zealand isolates, but only 85% identical to that of WClMV-M, the other New Zealand isolate, because of heterogeneity in the C-termini of CP amino acid sequences. The implication of this CP heterogeneity is discussed. Received 30 August 2001/ Accepted in revised form 11 January 2002