Detection and subgrouping of Cucumber mosaic virus isolates by TAS-ELISA and immunocapture RT-PCR

Eight mouse hybridoma cell lines secreting monoclonal antibodies (MAbs) against Cucumber mosaic virus (CMV) were produced. Analysis of the specificities of the MAbs against CMV isolates by triple antibody sandwich (TAS)-ELISA demonstrated that four MAbs were specific for subgroup I (S-I) isolates and two for subgroup II (S-II) isolates, whereas another two MAbs could detect both S-I and S-II isolates. TAS-ELISA and immunocapture RT-PCR (IC-RT-PCR) methods were then established for reliable and efficient detection and subgrouping of CMV isolates using the produced MAbs. When 197 field samples collected from six provinces in China were tested by TAS-ELISA, 130 samples were found to be infected by CMV. Among them, 121 samples were infected by S-I isolates (93.1%) and another nine samples by S-II isolates (6.9%). In IC-RT-PCR using the MAbs and specific primers in the region of the coat protein (CP) gene, samples shown to contain S-I isolates by TAS-ELISA gave one specific band about 500 nucleotides in length, whereas samples containing S-II isolates produced a single band with the length of approximately 600 nucleotides. The validity and reliability of the results of TAS-ELISA and IC-RT-PCR was confirmed by sequencing and phylogenetic analysis of nearly full-length CP genes of the isolates.     

Purification and partial characterization of sugar beet yellows virus

A technique was devised for obtaining purified infectious preparations of sugar beet yellows virus (SBYV) particles. As the use of mechanical blending resulted in shearing of particles, infected leaves were hand ground in a mortar; the extracts were clarified with bentonite, concentrated using polyethylene glycol, and further purified by rate zonal and isopycnic gradient centrifugation. The purified particles had a modal length of 1270 nm, sedimented at 110 S, had an unusual ultraviolet absorption spectrum and contained about 5% RNA. The molecular weight of the infectious RNA, estimated by sedimentation and by gel electrophoresis was about 4.3 × 10⁶; that of the coat protein subunit was about 23,500. These properties are discussed in relation to the structure of the virus particles and also are compared with the properties of viruses allied to SBYV.     

The protein and nucleic acid of beet yellows virus

By analysis on SDS-polyacrylamide gels, the coat protein of beet yellows virus was shown to consist of a single polypeptide of molecular weight about 22,400. The amino acid composition and tryptic peptide map also indicate a molecular size close to 22,000. Viral RNA comprising 6% by weight of the particles is single stranded and has the base composition U, 23%; C, 22%; A, 27%; G, 28%. Its size, estimated by sedimentation and gel electrophoresis, is close to 4.6 × 10⁶. We propose that in the virus particle the single helical RNA strand is embedded at a radius of 3.3 nm in a protein coat comprised of approximately 3400 protein subunits, giving a total particle weight of 80 million daltons.     

The detection of beet western yellows virus and beet mild yellowing virus in crop plants using the polymerase chain reaction.

Oligonucleotide primers were synthesised corresponding to conserved sequences between three isolates of beet western yellows virus (BWYV), flanking a 913 base fragment of BWYV genomic RNA. Using the polymerase chain reaction (PCR), these primers successfully amplified the target fragment in total RNA extracts from two oilseed rape plants infected with different isolates of BWYV. The PCR products were readily detected by staining with ethidium bromide following agarose gel electrophoresis, but the limit of detection could be increased further by Southern blotting. However, three isolates of beet mild yellowing virus (BMYV) in sugar beet did not give a signal which could be detected by ethidium bromide staining, although the target fragment could be detected by Southern blotting. The primers used have the potential to detect BWYV in crops with far greater sensitivity than enzyme-linked immunosorbent assay or nucleic acid hybridisation (dot-blotting) and may be capable of distinguishing between BWYV and BMYV. The application of PCR to detection and distinction of luteoviruses in general is discussed.     

Nucleotide sequence of cDNA encoding the coat protein of beet yellows virus

A cDNA clone of beet yellows viral RNA expressed the viral coat protein gene inE. coli. The sequence of the 2724 nucleotide insert revealed three open reading frames, the 3 of which was shown to be the coat protein cistron. This cistron is expressed inE. coli, in spite of there being no obvious ribosome binding site upstream.     

Maximizing the detection capability of a beet western yellows virus ELISA system

Conditions for maximizing detection of a California isolate of beet western yellows virus (BWYV) were investigated with the double-sandwich enzyme-linked immunosorbent assay (ELISA) system. Within-plate variability was found to account for less than 1% of the total variation observed on individual microtiter plates. Variability across plates was greater than within plates and accounted for less than 10% of the total variation. Significant reductions in absorbance were observed when either coating or conjugated IgGs were incubated for 2 h at 37 degrees C. Incubation of coating IgGs overnight at 4 or 22 degrees C gave good results. No differences in absorbance were observed when antigen sources were incubated at 37, 22, or 4 degrees C. Absorbance at 1, 3 or 6 h incubation of conjugated IgGs at 4 or 22 degrees C was low to moderate but after 18 or 24 h absorbance was markedly increased without increasing background. Efficient extraction of virus from host and vector tissues increased absorbance. Carborundum added to the extraction buffer worked well when tissue samples were ground in a mortar and pestle but highest absorbance resulted when test samples were prepared with a high-speed tissue homogenizer. Background levels were unacceptably high when 10 or more Myzus persicae were pooled for analysis. Since virus can be detected in 3 or even 1 aphid, pooling for detection is unnecessary. Optimization of the BWYV ELISA system made possible the detection of virus quantities less than 2 ng/ml.     

Glycosylation of beet western yellows virus proteins is implicated in the aphid transmission of the virus

Summary. Beet western yellows virus relies on the aphid M. persicae for its transmission in a persistent and circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary gland epithelial barriers by a transcytosis mechanism where vector receptors interact with virions. The aphid and the peptidic viral determinants implicated in this interaction mechanism have been studied. In this paper, we report that the coat and the readthrough proteins that constitute the capsid of this virus are glycosylated. Modification of the glucidic core of these structural viral proteins by oxidation with sodium metaperiodate or deglycosylation with N-glycosidase F or α-D-galactosidase abrogates the aphid transmission of the virus. Aphid transmission could also be inhibited by lectins directed against α-D-galactose when aphids were allowed to acquire virus on artificial membranes. These results suggest that the glucidic cores of the capsid proteins of beet western yellows virus contain α-D-galactose residues that are implicated in virus-aphid interaction and promote aphid transmission of the virus.     

Detection of bovine respiratory syncytial virus by RT-PCR

The paper presents the results of studying the diagnostic efficiency of RT-PCR for the detection of respiratory syncytial virus in cattle of different ages. Glycoprotein F gene sequences were used as a target for amplification. The sensitivity of the reaction was 10 TCD50/ml and the virus detection rate in biomaterials averaged 19%. samples. That in RT-PCR correlated with the presence of clinical signs in sick animals.     

Biochemical and serological comparisons between carnation yellow fleck virus and sugar beet yellows virus protein subunits.

Carnation yellow fleck virus is serologically related to sugar beet yellow virus. Amino acid analyses and tryptic peptide maps indicate about 50 amino acid differences between the proteins from the two viruses.     

Detection of canine distemper virus in dogs by real-time RT-PCR

Canine distemper virus is the etiological agent of a severe disease in dogs and many other carnivores. Clinical diagnosis of canine distemper is difficult due to the broad spectrum of signs that may be confounded with other respiratory and enteric diseases of dogs. Accordingly, a laboratory confirmation is required for suspected cases. In this study a real-time RT-PCR assay was developed for detection and quantitation of canine distemper virus. The assay exhibited high specificity as all the negative controls (no-template-controls and samples from healthy sero-negative dogs) and other canine pathogens were not misdetected. Up to 1 x 10(2) copies of RNA were detected by the TaqMan assay, thus revealing a high sensitivity. Quantitative TaqMan was validated on clinical samples, including various tissues and organs collected from dogs naturally infected by canine distemper virus. Urines, tonsil, conjunctival swabs and whole blood were found to contain high virus loads and therefore proved to be suitable targets for detection of canine distemper virus RNA.     

Detection of alien chromatin conferring resistance to the beet cyst nematode (Heterodera schachtii Schm.) in cultivated beet (Beta vulgaris L.) using in situ hybridization

Chromatin originating from wild beets of the genus Beta, section Procumbentes, has been investigated in nematode-resistant hybrid-derived lines of sugar beet (Beta vulgaris L.) by in situ hybridization using satellite, telomeric and ribosomal DNA repeats, a yeast artificial chromosome (YAC) and total genomic DNA as probes. The alien chromosome was detected in three monosomic addition lines(2n=18+1) by genomic in situ hybridization. Fluorescence in situ hybridization with a genome-specific satellite repeat and YAC DNA enabled the visualization of Procumbentes chromosomes, and in double-target hybridization it was shown that they do not carry 18S–5.8S–25S rRNA and 5S rRNA genes. The wild beet-specific satellite repeat and the telomere sequence from Arabidopsis thaliana were used to perform a structural analysis of the wild beet chromosome fragments of two resistant fragment addition lines. It was shown that one physical end of the chromosome fragments consists of telomeric repeats. Comparison of fragment sizes indicated that the small chromosome fragments harbouring the resistance gene most likely resulted from the loss of one wild beet chromosome arm and an internal deletion of the remaining arm.This revised version was published online in November 2005 with corrections to the Cover Date.     

Detection of Tomato black ring virus by real-time one-step RT-PCR

A TaqMan-based real-time one-step RT-PCR assay was developed for the rapid detection of Tomato black ring virus (TBRV), a significant plant pathogen which infects a wide range of economically important crops. Primers and a probe were designed against existing genomic sequences to amplify a 72 bp fragment from RNA-2. The assay amplified all isolates of TBRV tested, but no amplification was observed from the RNA of other nepovirus species or healthy host plants. The detection limit of the assay was estimated to be around nine copies of the TBRV target region in total RNA. A comparison with conventional RT-PCR and ELISA, indicated that ELISA, the current standard test method, lacked specificity and reacted to all nepovirus species tested, while conventional RT-PCR was approximately ten-fold less sensitive than the real-time RT-PCR assay. Finally, the real-time RT-PCR assay was tested using five different RT-PCR reagent kits and was found to be robust and reliable, with no significant differences in sensitivity being found. The development of this rapid assay should aid in quarantine and post-border surveys for regulatory agencies.     

逆转录-聚合酶链反应方法检测乙型脑炎病人标本

目的 逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）检测乙型脑炎（乙脑）病人标本方法的建立和评估。方法 建立ＲＴ－ＰＣＲ法,了解该方法用于乙脑病毒检测的敏感性,特异性,并用于临床疑似乙脑病人血清及脑脊液（ＣＳＦ）标本的检测,并与反向被动血凝抑制实验（ＲＰＨＩ）方法进行比较分析。结果 用该ＲＴ－ＰＣＲ法检测高顺生株（高株）敏感性可达６４ＰＦＵ。共检测临床疑似乙脑病人标本３８份,对ＣＳＦ中乙型脑炎病毒（ＪＥＶ）     

Subcellular localization of the HSP70-homolog encoded by beet yellows closterovirus.

Closteroviridae is the only viral family coding for a homolog of HSP70 (HSP70h). Polyclonal antiserum to recombinant beet yellows closterovirus (BYV) HSP70h was generated and used for immunogold labeling of the leaf samples derived from the infected Nicotiana benthamiana plants. Ultrastructural analysis revealed the preferential accumulation of BYV in phloem, although occasional infection of the leaf mesophyll cells was also observed. The strongest HSP70h-specific labeling was associated with virion aggregates and vesicles harboring scattered virions. HSP70h was also observed in close proximity of plasmodesmata and inside the plasmodesmatal channels. The possible role of the BYV HSP70h in RNA encapsidation was tested in tobacco protoplasts. A BYV mutant possessing an inactivated HSP70h gene exhibited no detectable encapsidation defects. Collectively, the obtained results suggested that closteroviral HSP70h escorts the virions to their destinations inside the infected cells and possibly participates in the intercellular translocation of BYV.