Application of Phage Display in Selecting Tomato spotted wilt virus-Specific Single-Chain Antibodies (scFvs) for Sensitive Diagnosis in ELISA

ABSTRACT A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S, which allowed the scFvs to be expressed as alkaline phosphatase fusion proteins, 17 different scFv antibodies were obtained. Of these, 12 scFvs were directed against the nucleoprotein (N) and 5, putatively, against the glycoproteins (G1 and G2). Five of the N-specific antibodies cross-reacted with two other tospoviruses (Tomato chlorotic spot virus and Groundnut ringspot virus), but none recognized the more distantly related tospoviruses Impatiens necrotic spot virus, Watermelon silverleaf mottle virus, Iris yellow spot virus, or Physalis severe mottle virus. The successful use of one of the antibodies as coating and detection reagent in a double-antibody sandwich enzyme-linked immunosorbent assay showed the potential of the phage display system in obtaining antibodies for routine TSWV diagnosis.     

Development of a recombinant antibody ELISA test for the detection of Polymyxa betae and its use in resistance screening

An ELISA test was developed for the quantitative detection of the obligate parasite Polymyxa betae , the vector of Beet necrotic yellow vein virus (BNYVV), in infected sugarbeet roots. The test used monoclonal and polyclonal antibodies raised to a recombinantly expressed glutathione-S-transferase (GST) from P. betae . A close correlation was found between the number of P. betae zoospores in serially diluted suspensions and absorbance values in the ELISA test. Time-course studies of plants grown in naturally infested soils in controlled environment tests demonstrated the value of the ELISA test in screening for P. betae resistance. In preliminary tests, P. betae -resistant accessions of the wild sea beet ( Beta vulgaris ssp. maritima ), which might be used to restrict the transmission of BNYVV, were identified.     

在我国发生的甜菜坏死黄脉病毒病

 1978年以来,在内蒙包头、呼和浩特等地发现了一种甜菜新病害。地上部表现叶片黄化、枯斑坏死、沿脉黄化坏死等症状;地下部表现次生侧根异常增生。根据鉴别寄主的症状,病株根部多粘菌(Polymyxa betae)的寄生和病毒形态及粒子长度分布特征确定内蒙所发现的这种新病害即国外报道的由Polymyxa betae传带的甜菜坏死黄脉病毒所致的甜菜丛根病。     

Generation and evaluation of movement protein‐specific single‐chain antibodies for delaying symptoms of Tomato spotted wilt virus infection in tobacco

This study investigated whether single‐chain antibodies (scFvs) specific for a viral movement protein could accumulate in the plant cell cytosol and restrict viral systemic infection in plants. Nine chicken scFv fragments against the Tomato spotted wilt virus (TSWV) movement protein (NS_M) were isolated by phage display. Soluble scFvs were produced in bacteria and the NS_M binding activity of purified scFvs was confirmed. The nine scFv genes were cloned into a plant expression vector enabling recombinant protein accumulation in the plant cell cytosol. Immunoblot analysis demonstrated that two of the nine chicken scFvs accumulated to high levels (5·9 and 8·0% of total soluble protein). Bioassays of viral infection using transgenic tobacco plants producing NS_M‐specific chicken scFvs showed delayed symptom development when compared to non‐transgenic control plants, indicating that expression of antibodies recognizing the TSWV movement protein is a potential strategy for generating resistant plants.     

Rhizomania disease of sugar beet in England

Rhizomania disease was first detected in the UK in 1987, in a single crop in Suffolk. Affected plants had pale leaves, often upright, narrow and rolled; roots were small, often with constrictions, warty outgrowths, proliferation of fibrous roots, and vascular staining. Disease occurred in strips at right angles to one another, parallel with directions of cultivation, suggesting that the previous beet crop had also been infected. Beet necrotic yellow vein virus was detected by ELISA, by electron microscopy, and by transmission to indicator species. It was sometimes associated with beet soil-borne virus. The affected crop was destroyed with herbicide. No other outbreaks were detected in subsequent surveys of crops in 1987.     

Viruses and virus-like pathogens transmitted by zoosporic fungi1

The viruses and virus-like pathogens transmitted by zoosporic fungi are reviewed. The nine furoviruses (and possible members of the group), with labile rod-shaped particles, have nearly all been shown to be transmitted by plasmodiophoromycete vectors. As they have been reviewed extensively elsewhere, they are covered only briefly; important examples are beet necrotic yellow vein furovirus and potato mop-top furovirus. Five viruses with filamentous particles, tentatively recognized as poty viruses, are transmitted by Polymyxa graminis. Within this group, wheat yellow mosaic virus should be considered to include wheat spindle streak mosaic virus, while the M and NM forms of barley yellow mosaic virus, the best known members of the group, should probably be regarded as distinct viruses. Chytrids (especially Olpidium brassicae) transmit a variety of viruses in different groups (e.g. tobacco necrosis necrovirus, lettuce big-vein virus, melon necrotic spot carmovirus, red clover necrotic mosaic dianthovirus). Finally, several diseases caused by uncharacterized pathogens appear to be transmitted by O. brassicae: freesia leaf necrosis, lettuce ring necrosis, pepper yellow vein, watercress chlorotic leaf spot.     

Serotypes of beet soil-borne furovirus from FRG and Sweden1

Four virus isolates were obtained in FRG and Sweden from sugarbeet roots which had become infected via the soil. All isolates had rod-shaped particles of four or five different lengths. Serologically they were unrelated to beet necrotic yellow vein furovirus and several other viruses of a similar morphology including two serologically distinct sugarbeet isolates from California (US). Our isolates were, however, serologically related to beet soil-borne furovirus which has been described in England. They could be subdivided into two serotypes.     

Variation in Pathogenicity and Multiplication of Beet Necrotic Yellow Vein Virus (BNYVV) in Relation to the Resistance of Sugar-beet Cultivars

Some partially resistant cultivars varied in their response to beet necrotic yellow vein virus (BNYW), which could be due to the occurrence of different pathotypes. In the past three different types of BNYVV could be identified. Since in the field no consistent cultivar×virus source interaction could be detected, greenhouse trials were carried out under more standardised conditions, starting with a similar initial density of BNYVV. Cultivars with different degrees of resistance varied in their response to various types of beet necrotic yellow vein virus (BNYVV). The B type appeared to be less damaging than the A and P types. The virus content in the tap roots and the ratio of the virus content in tap roots to that in lateral roots were both higher in P type than in A or B type infections indicating that the P type moves more rapidly in the plants than the two other BNYVV types. The percentage of plants in which the virus reaches only a low concentration (less than 56ng/ml of sap) is much lower in P type than in A or B type infections. Frequency distribution diagrams of individual plants showing different BNYVV levels reveal considerable differences between various cultivars.     

The role of alternative hosts of Polymyxa betae in transmission of beet necrotic yellow vein virus (BNYVV) in England

The host range of beet necrotic yellow vein virus (BNYVV) and Polymyxa betae was determined by growing plants in naturally infested soils from rhizomania outbreaks in England. Apart from Beta vulgaris , plant species infected by BNYVV were included in the families Chenopodiaceae ( Atriplex patula, Chenopodium bonus-henricus, C. hybridum, C. polyspermum and Spinacia oleracea ), Amaranthaceae ( Amaranthus retroflexus ) and Caryophyllaceae ( Silene alba, S. vulgaris, S. noctiflora and Stellaria graminea ). Only P. betae isolates from B. vulgaris, C. polyspermum and S. oleracea were found to be able to transmit BNYVV back to sugar beet. When a range of weed plants from infected fields were tested, none were found to be infected by BNYVV. Therefore, it seems likely that the weed hosts play only a minor role in the spread of rhizomania disease compared to that of sugar beet, other Beta vulgaris crop types or spinach.     

Horizontal spread of beet necrotic yellow vein virus in soil

Horizontal dispersal of beet necrotic yellow vein virus (BNYVV) by means of viruliferous zoospores ofPolymyxa betae was studied in greenhouse experiments. BNYVV was not detected in roots of sugar beet plants grown in silver sand for 4 weeks at a root-free distance of 5 cm from eitherP. betae- and BNYVV-infected plants or BNYVV-infested soil. Spread of BNYVV from inoculum sources in the field was studied in the absence and presence of tillage practices. Active dispersal in combination with root growth from and towards point sources of inoculum contributed only little to horizontal dispersal of viruliferous inoculum and spread of disease during the season, as determined for one soil type, two different years and in the absence of tillage and tread. In the second beet crop after application of inoculum to whole field plots, more BNYVV-infected plants were detected at 2 m than at 8 m distance from the infested plots in the tillage direction. In the third year, disease incidence at 8 m was high and equivalent to that at 2 m.     

Partial nucleotide sequence analysis of Turkish isolates of Beet necrotic yellow vein virus (BNYVV) RNA-3

A survey was carried out to detect Beet necrotic yellow vein virus (BNYVV) in soil samples using RT-PCR and bait plant techniques from the sugar beet production area of Tokat, Turkey in 2001. More than 80% of the soil samples analyzed were found to be contaminated with the virus. The partial nucleotide sequence of cDNA corresponding to RNA-3 of BNYVV isolates were analyzed for six different regions of Tokat province. All isolates were assigned to type A strains based on RFLP analysis and DNA sequences. Sequence comparison revealed differences at amino acid positions 35, 68, 71 and 179 of the P25 coding region amongst Turkish isolates. Additionally, all Turkish isolates were compared with Japanese, French, Kazakh, Italian and Belgian isolates.     

Selection of single-chain variable fragment antibodies to black currant reversion associated virus from a synthetic phage display library

ABSTRACT Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.     

Polymyxa betae zoospores as vectors of beet necrotic yellow vein furovirus1

When beet seedlings exposed as bait plants in infested soil were placed in a nutrient solution, they released Polymyxa betae zoospores, infected with beet necrotic yellow vein furovirus. The roots produced the first zoospores 5 days after the start of soil baiting. When seedlings were inoculated with zoospore suspensions, infection occurred within 5 min and reached a maximum in 30 min. The suspensions remained infectious for at least 2 h after removal of the bait plants from which the zoospores were released. So many spores were released into the suspension that disease transmission could be obtained within half an hour from an infected plant to a healthy plant, placed together into fresh medium. Suspensions could be diluted 1/16 with nutrient solution without any loss of infectivity, whereas 1/4 dilution with tap water resulted in a complete loss of infectivity.     

Effect of sugar beet cultivars with different levels of resistance to beet necrotic yellow vein virus on transmission of virus byPolymyxa betae

The effect of resistance of sugar beet cultivars to beet necrotic yellow vein virus (BNYVV) on virus content of resting spore clusters of the vectorPolymyxa betae was studied in controlled environments and in naturally infested fields. The total number of resting spore clusters formed in roots of a partially resistant and a susceptible cultivar did not differ when assessed 6 and 12 weeks after inoculation with viruliferous resting spores. Transmission experiments showed that in partially resistant plants, having a low virus content in the roots, the population of resting spores formed was less viruliferous than that in susceptible plants with a high virus content. Consequently, growing a resistant cultivar can be expected to delay the build-up of virus inoculum in soil.In a trial field sampled in 1991, the inoculum potential of BNYVV (most probable number of viruliferousP. betae propagules) in soil was lower after growing a partially resistant cultivar than after growing a susceptible one. On the other hand, in four sites sampled in 1990, inoculum potential in soil was hardly increased by growing sugar beet and was not significantly affected by the cultivar grown.     

Resistance against rhizomania disease via RNA silencing in sugar beet

Rhizomania is one of the most damaging and widely spread diseases in major sugar beet growing regions of the world. The causal agent, beet necrotic yellow vein virus (BNYVV), is transmitted via the fungus Polymyxa betae, which retains it in the field for years. In this study, an RNA silencing mechanism was employed to induce resistance against rhizomania using intron‐hairpin RNA (ihpRNA) constructs. These constructs were based on sequences of the BNYVV 5′‐untranslated region of RNA‐2 or the flanking sequence encoding P21 coat protein, with different lengths and orientations. Both transient and stable transformation methods produced effective resistance against rhizomania correlated with the transgene presence. Among the constructs, those generating ihpRNA structures with small intronic loops produced the highest frequencies of resistant events. The inheritance of transgenes and resistance was confirmed over generations in stably transformed plants.     

First Record of A and B Type Beet necrotic yellow vein virus in Sugar Beets in Sweden

Natural infections of sugar beet with Beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania, have been detected for the first time in Sweden in two small areas, one on the island of Öland and one in the Southeastern part of Scania. Single strand conformation polymorphism analyses of PCR products revealed that the infections on Öland were produced by A type BNYVV, whereas those in Scania were caused by the B type. This suggests that BNYVV has been introduced into Sweden at least twice. Alternatively, the virus may have invaded sugar beet from unknown native hosts. BNYVV RNA 5 was not detected in the samples investigated.     

Differential abundance of proteins in response to Beet necrotic yellow vein virus during compatible and incompatible interactions in sugar beet containing Rz1 or Rz2

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is an important disease affecting sugar beet. Control is achieved through planting of resistant varieties; however, following the introduction of Rz1, new pathotypes that overcome resistance have appeared. To understand how BNYVV overcomes resistance, we examined quantitative protein differences during compatible and incompatible interactions when sugar beet is infected with either a traditional A-type strain or with an Rz1 resistance breaking strain. Proteomic data suggest distinct biochemical pathways are induced during compatible and incompatible sugar beet interactions with BNYVV. Pathways including the detoxification of reactive oxygen species, UB/proteasome, and photosynthesis should be studied in more depth to characterize roles in symptom development.     

Analysis of the resistance-breaking ability of different beet necrotic yellow vein virus isolates loaded into a single Polymyxa betae population in soil

The genome of most Beet necrotic yellow vein virus (BNYVV) isolates is comprised of four RNAs. The ability of certain isolates to overcome Rz1-mediated resistance in sugar beet grown in the United States and Europe is associated with point mutations in the pathogenicity factor P25. When the virus is inoculated mechanically into sugar beet roots at high density, the ability depends on an alanine to valine substitution at P25 position 67. Increased aggressiveness is shown by BNYVV P type isolates, which carry an additional RNA species that encodes a second pathogenicity factor, P26. Direct comparison of aggressive isolates transmitted by the vector, Polymyxa betae, has been impossible due to varying population densities of the vector and other soilborne pathogens that interfere with BNYVV infection. Mechanical root inoculation and subsequent cultivation in soil that carried a virus-free P. betae population was used to load P. betae with three BNYVV isolates: a European A type isolate, an American A type isolate, and a P type isolate. Resistance tests demonstrated that changes in viral aggressiveness towards Rz1 cultivars were independent of the vector population. This method can be applied to the study of the synergism of BNYVV with other P. betae-transmitted viruses.     

Fusion proteins of single-chain variable fragments derived from phage display libraries are effective reagents for routine diagnosis of potato leafroll virus infection in potato

ABSTRACT A panel of 11 different single-chain variable fragment antibodies (scFv) that bind to potato leafroll virus (PLRV) has been studied to assess each one's suitability as practical diagnostic tools. The scFv, previously obtained from naive phage display libraries, were expressed in Escherichia coli as fusion proteins. The fusion proteins comprised scFv joined to either the human light chain kappa constant domain (C(L)), an amphipathic helix (Zip), a combination of C(L) and Zip, or alkaline phosphatase (AP/S). The fusion proteins were tested for their ability to detect, or trap on enzymelinked immunosorbent assay (ELISA) plates, PLRV in extracts of infected potato leaves. The tests done with the different scFv fusion proteins were compared with a standard triple-antibody sandwich (TAS)-ELISA that employs a rabbit polyclonal antibody preparation to coat microtiter plates and a monoclonal antibody, SCR3, to detect PLRV. Of 11 scFvC(L) fusion proteins, 7 detected PLRV as readily as SCR3 when used as detecting antibodies in TAS-ELISA. The limit of detection of purified PLRV for the different scFvC(L) fusion proteins ranged from 250 to 5 ng/ml; that for SCR3 is 5 ng/ml. Of the 11 scFv, 4 cross-reacted with some other luteoviruses. Several scFvC(L) and scFvC(L)Zip fusion proteins trapped PLRV from extracts of infected potato leaves as effectively as the polyclonal antibody preparation. Four scFv fusion proteins were used in a stem print assay to detect PLRV, and the results were similar to those obtained in tests using SCR3. The scFvC(L) fusion proteins retained activity for at least 6 months at 4 degrees C, and all scFv fusion proteins were fully active on reconstitution after lyophilization. A fully recombinant ELISA was devised that detected PLRV in extracts of infected potato, with results comparable to those obtained using the standard TAS-ELISA. The advantages of using scFv fusion proteins for the routine detection of plant viruses include the ability to produce large quantities of reagents cheaply in bacterial fermenters and to incorporate them into standardized tests.     

The Effect of Different Levels of Beet Cyst Nematodes (Heterodera schachtii) and Beet Necrotic Yellow Vein Virus (BNYVV) on Single and Double Resistant Sugar Beet Cultivars

Beet cyst nematode-resistant cultivars, which were introduced recently, originated from the homozygous inbred line B883. This translocation product was unstable and the transmission of resistance when crossed with a susceptible cultivar did not exceed 94%. Tests with the resistant cultivars in climate cabinets showed a wide variety of resistance against Heterodera schachtii and beet necrotic yellow vein virus (BNYVV), expressed as average numbers of infective units per plant and percentages of resistant plants. In a series of field trials at different levels of infection of H. schachtii, their multiplication rates on all resistant cultivars depended on the initial density, which was caused by the presence of small numbers of susceptible plants. Since tolerance to wilting was also incorporated in B883, reasonable yields were obtained in the presence of H. schachtii. However, at increasing initial densities of H. schachtii, yields decreased considerably, since penetrating juveniles cause a hypersensitivity reaction in resistant plants. Based upon the results of three series of field trials, it was concluded that resistant cultivars should preferably be applied at population densities between 500 and 2000 eggs and juveniles of H. schachtii per 100ml of soil. Cultivars with double resistance against H. schachtii and BNYVV behaved like those with H. schachtii resistance in soils infected with beet cyst nematodes, but not with BNYVV. In soils with a combined infection of H. schachtii and BNYVV double resistant cultivars were far superior to single resistant ones, since damage caused by BNYVV was far more serious than damage caused by H. schachtii. No substantial interaction between soil pathogens nor types of resistance could be detected.