Significance of the heterologous encapsidation of zucchini yellow mosaic potyvirus in transgenic plants expressing plum pox potyvirus capsid protein1

Transgenic Nicotiana benthamiana plants expressing the coat protein of an aphid-transmissible strain of plum pox potyvirus (PPV-D) were infected with an aphid non-transmissible strain of another potyvirus, zucchini yellow mosaic potyvirus (ZYMV-NAT). Non-viruliferous Myzus persicae could acquire and transmit ZYMV-NAT from these plants but not from infected N. benthamiana control plants (not transformed, or transformed by the vector alone). Immunosorbent electron microscopy experiments using the decoration technique revealed that ZYMV-NAT virus particles in the infected transgenic plants expressing the PPV coat protein could be coated not only with ZYMV antibodies but also, on segments of the particles, with PPV antibodies. This suggests that aphid transmission of ZYMV-NAT occurred through heterologous encapsidation, and reveals a potential risk of releasing genetically engineered plants expressing viral coat proteins into the environment.     

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.     

Transmission by aphids of potato spindle tuber viroid encapsidated by potato leafroll luteovirus particles

Potato spindle tuber viroid (PSTVd) was transmitted by Myzus persicae to Physalis floridana from P. floridana plants that also were infected with potato leafroll luteovirus (PLRV), whereas it was not transmitted by aphids from plants infected with PSTVd alone. Dot-blot hybridisation was used to detect PSTVd. The results indicate that PLRV can assist PSTVd in its transmission by M. persicae. Doubly infected, aphid-inoculated P. floridana plants from the previous experiment were used as the source plants in aphid transmission tests to the tomato cv. Rutgers, P. floridana and Datura stramonium. PSTVd was detected in 17 of 30 plants of tomato. The viroid was not detected by dot-blotting in any plant of P. floridana and D. stramonium in this experiment, but it was recovered from some plants by sap inoculation of the Rutgers plants. Treatment with RNase A of PLRV preparations purified from doubly infected plants indicated that PSTVd was encapsidated by PLRV particles.     

Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe. M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe.M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

Effects of Cry1Ac toxin of Bacillus thuringiensis and nuclear polyhedrosis virus of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on larval mortality and pupation

In the laboratory, the percentage mortality and pupation of Helicoverpa armigera (Hübner) were investigated when larvae were exposed to Cry1Ac of Bacillus thuringiensis Berliner, nuclear polyhedrosis virus of H. armigera (HaNPV) or Cry1Ac and HaNPV together. The results revealed that interactions between Cry1Ac and HaNPV varied with bioassay method and concentration of the suspension. When larvae were infected using a suspension containing both HaNPV and Cry1Ac, most combinations of Cry1Ac (62.5, 125 and 250 microg mL(-1)) and HaNPV (1.2 x 10(6), 6.0 x 10(6) and 3.0 x 10(7) PIB mL(-1)) showed an antagonistic effect. In the bioassay procedure where larvae were force fed diet containing Cry1Ac 48 h after being infected by HaNPV, interaction between Cry1Ac (0.5, 1, 2, 4 and 8 microg mL(-1)) and HaNPV (6.0 x 10(6) and 3.0 x 10(7) PIB mL(-1)) showed an additive effect, while combinations of Cry1Ac (0.5, 1, 2 and 4 microg mL(-1)) and HaNPV (1.2 x 10(6) PIB mL(-1)) showed an antagonistic effect. In the bioassay procedure where larvae being infected by HaNPV were fed on Cry1Ac diet from neonate to death or pupation, the results suggested that Cry1Ac and HaNPV showed an additive interaction. The percentage mortality was lower in the treatment of larvae infected by transgenic Bt cotton leaf discs containing HaNPV suspension than in the treatment of larvae by conventional cotton leaf discs containing HaNPV, while the pupation rate was higher. The combination of Bt cotton and HaNPV showed antagonism. The present results showed that a combination of Cry1Ac and HaNPV usually resulted in mortality levels greater than in the case of Cry1Ac but not greater than with the virus alone.     

马铃薯种苗复合感染病毒多重RT-PCR同步快速检测

 基于马铃薯病毒ssRNA的快速制备方法,根据马铃薯病毒CP基因序列设计PVX、PVS、PVY和PLRV特异性引物对、P1基因序列设计PVA特异性引物对,建立了多重RT PCR快速检测体系,可以同步检出复合侵染马铃薯种苗主要病毒,灵敏度比传统的ELISA至少高100倍。结合生物活性稳定剂研制的固相化检测试剂盒,已用于四川和重庆等15个县市田间与苗圃248个马铃薯显症或无症样品的实际检测,表明四川和重庆地区2~3种马铃薯病毒往往复合侵染(PVX、PVA和PVS三种病毒复合侵染或PLRV和PVY二种病毒复合侵染)。     

Hairy nightshade as a potential Potato leafroll virus (Luteoviridae: Polerovirus) inoculum source in Pacific Northwest potato ecosystems

Hairy nightshade, Solanum sarrachoides, is a solanaceous weed found abundantly in Pacific Northwest potato ecosystems. It serves as a reservoir for one of the important potato viruses, Potato leafroll virus (PLRV) (Luteoviridae: Polerovirus), and its most important vector, the green peach aphid, Myzus persicae (Homoptera: Aphididae). Laboratory research indicated an increased green peach aphid settling and performance on S. sarrachoides than on potato. It also revealed that green peach aphids transmitted PLRV more efficiently from S. sarrachoides to potato than from potato to potato. To test the efficiency of S. sarrachoides as an inoculum source in the field, a two season (2004 and 2005) trial was conducted at Kimberly, Idaho. Two inoculum sources, PLRV-infected potato and PLRV-infected S. sarrachoides, were compared in this trial. Green peach aphid density and temporal and spatial PLRV spread were monitored at weekly intervals. Higher densities of green peach aphids were observed on plots with S. sarrachoides and inoculum sources (PLRV-infected S. sarrachoides and potato) than on plots without S. sarrachoides and inoculum sources. PLRV infection in plots with PLRV-infected S. sarrachoides was similar to or slightly higher than in plots with PLRV-infected potato as an inoculum source. Temporal and spatial PLRV spread was similar in plots with either inoculum source. Thus, S. sarrachoides is as efficient as or a better PLRV inoculum source than potato.     

Occurrence of potyvirus and potexvirus infections in black bryony (Tamus communis) in Devon, UK

Leaf samples of black bryony (Tamus communis L.) from Devon, UK, showing various virus-like symptoms contained potyvirus-like particles (normal length c. 790 nm) and cytoplasmic cylindrical (pinwheel) inclusions. In immunoelectron microscopy, particles of most samples reacted with antiserum to dioscorea greenbanding mosaic virus, a potyvirus isolate from Dioscorea rotundata in Togo which is related to yam mosaic virus from the Ivory Coast. Potyvirus particles were not transmitted by sap or aphids (Myzuspersicae) from infected black bryony to black bryony seedlings or Nicotiana benthamiana. One sample from a symptomless plant of black bryony contained a potexvirus which formed massed virion aggregates in the cytoplasm of cells of black bryony, Nicotiana benthamiana and N. megalosiphon. Virions of the potexvirus (normal length 553 nm) contained a coat protein with an apparent molecular weight of 27.7 kd. The potexvirus differed from an Italian potexvirus isolate from black bryony by a serological differentiation index of 4 and gave only weak or no reactions with 23 other antisera to potexviruses, including dioscorea latent virus. The potexvirus caused systemic symptoms in only a few host plants and could be transmitted back to black bryony in which it caused no symptoms. It is provisionally named tamus latent virus.     

甘肃省马铃薯主要病毒病发生情况调查

2015年-2016年,在甘肃省10个地市24个马铃薯主栽县(区)146个生态区域(乡镇)采集了757份具有典型症状的马铃薯样品,应用DAS-ELISA法进行检测,筛查6种主要病毒(PVX、PVY、PLRV、PVA、PVS和PVM)。结果表明:631份样品检测到病毒,PVS的检出率最高,达47.03%,PVY次之,为33.82%,PVA最低,只有0.63%;发生复合侵染的病毒主要为PVY+PVS,复合侵染率达到10.13%,三种病毒复合侵染主要是PVY+PVS+PVM;病毒种类和感病程度与品种、地域有关。     

一种快速简便检测马铃薯病毒的方法——病毒细菌协同凝集法(VBA)的研究

在国内、外首次选用金黄色葡萄球菌(Staphylococc aureus)的No.180菌株用于病毒细菌协同凝集试验(Virobacterial agglutination test简称VBA)检测了来自马铃薯的4种不同形态粒子的病毒:马铃薯X病毒(PVX)、马铃薯丫病毒(PVY)、烟草花叶病毒(TMV)和马铃薯卷叶病毒(PLRV),其灵敏度达2.7～6.1ng/ml,检出病汁液的最大稀释度达10~4～10~5(PLRV1:500),较国内、外采用的Cown I菌株的灵敏度提高5～10倍。与血清学方法相比,VBA在2～3分种内就可获得结果,无假阳性反应,其灵敏度显著高于间接酶联法和免疫电镜,而接近于A蛋白酶联法。经抗血清致敏的菌体在4℃下保存4个月,对VBA检测的灵敏度没有影响。用VBA对采自田间的93个马铃薯病样进行检测,它们大多受2～3种病毒(PVX、PVY及PLRV)的复合侵染;和用间接酶联法及鉴别寄主检测的结果趋向一致。室内和田间试验均表明VBA灵敏度高、特异性强、快速简便和经济,尤其适合在基层单位中推广应用。     

Helper component mutations in nonconserved residues associated with aphid transmission efficiency of a pepper isolate of potato virus y

ABSTRACT The aphid transmission properties of a pepper isolate of potato virus Y belonging to the pathotype 1-2 (PVY 1-2) have been characterized. PVY 1-2 was not transmitted in plant-to-plant experiments, although purified virus particles were efficiently transmitted when supplemented with heterologous helper component (HC) of the transmissible isolate PVY 0 AT through membrane acquisition assays, indicating that its coat protein was functional in transmission. Additionally, virions of PVY 1-2 were able to bind to different HCs in in vitro binding assays. Analysis of the sequence of the PVY 1-2 HC gene and comparison with that of PVY 0 AT revealed 19 nucleotide differences, but only 2 resulted in amino acid changes, one of which induced a change of charge. Neither of these two amino acid changes occurred within the cysteine-rich domain, nor did they coincide with conserved motifs of the HC protein known to be involved in aphid transmission and which are present in all known potyvi-ruses. However, both changes are located in positions highly conserved among PVY strains. The possible role of both mutations on the activity of the PVY 1-2 HC in aphid transmission is discussed.     

A new aubergine disease caused by a whitefly‐borne strain of Tomato mild mottle virus (TomMMoV)

The aims of the present study were to further characterize the causal agent of a new viral disease of aubergines in Israel, first observed in 2003 and tentatively named eggplant mild leaf mottle virus (EMLMV) in a previous work, and to identify the vector responsible for its spread. The disease could be transmitted mechanically from infected source plants to healthy aubergines or laboratory test plants. Transmission electron microscopy (TEM) analysis of purified virus preparations indicated the presence of viral particles with a flexible filamentous morphology (approximately 720 nm long). TEM analysis of ultrathin sections prepared from infected leaf tissue revealed the presence of cytoplasmic inclusion bodies with pinwheel and crystalline structures, typical of those induced by potyviral infection. The viral coat protein subunit was shown to have a molecular weight of 37·5 kDa by SDS‐PAGE analysis. The viral particles reacted positively in western blot analysis with an antiserum against Tomato mild mottle virus (TomMMoV) from Yemen, described as a potyvirus, vectored by the aphid Myzus persicae. The current study describes some biological properties of EMLMV and presents evidence for its transmission by the whitefly Bemisia tabaci, but not by three aphid species. The taxonomic relationship between EMLMV and TomMMoV is discussed based on their biological characteristics and sequence analysis of their genomes. It is suggested that the Israeli EMLMV should be considered a distant strain of TomMMoV, designated TomMMoV‐IL, according to the present rules of Potyviridae molecular taxonomy.     

The N-Terminal Region of the Readthrough Domain Is Closely Related to Aphid Vector Specificity of Soybean dwarf virus

ABSTRACT It has been speculated that the N-terminal half of the readthrough domain (RTD) encoded by open reading frame 5 of Soybean dwarf virus (SbDV) is related to the vector specificity. To further investigate this hypothesis, transmissibility via aphids was tested on 17 SbDV isolates and comparisons of the deduced amino acid sequences of the coat protein (CP) and other proteins encoded by the RTD were made between these isolates. Isolates were distinguished into four strains: YS, causing yellowing in soybean and transmittable by Aulacorthum solani; DS, causing dwarfing and transmittable by A. solani; YP, causing yellowing and transmittable by Acyrthosiphon pisum; and DP, causing dwarfing and transmittable by A. pisum. Phylogenetic analysis showed that the trees for the CP and the C-terminal half of the RTD sequences contained clusters of isolates of the same symptom type, whereas the tree for the N-terminal half of the RTD contained clusters of isolates of the same aphid vector type. These results agreed with our previous data of the complete nucleotide sequences of four SbDV isolates, and strongly indicated a close relationship between the N-terminal half of the RTD amino acid sequences and aphid transmission specificity of SbDV.     

Plant and insect virus-like particles: emerging nanoparticles for agricultural pest management

Virus-like particles (VLPs) represent a biodegradable, biocompatible nanomaterial made from viral coat proteins that can improve the delivery of antigens, drugs, nucleic acids, and other substances, with most applications in human and veterinary medicine. Regarding agricultural viruses, many insect and plant virus coat proteins have been shown to assemble into VLPs accurately. In addition, some plant virus-based VLPs have been used in medical studies. However, to our knowledge, the potential application of plant/insect virus-based VLPs in agriculture remains largely underexplored. This review focuses on why and how to engineer coat proteins of plant/insect viruses as functionalized VLPs, and how to exploit VLPs in agricultural pest control. The first part of the review describes four different engineering strategies for loading cargo at the inner or the outer surface of VLPs depending on the type of cargo and purpose. Second, the literature on plant and insect viruses the coat proteins of which have been confirmed to self-assemble into VLPs is reviewed. These VLPs are good candidates for developing VLP-based agricultural pest control strategies. Lastly, the concepts of plant/insect virus-based VLPs for delivering insecticidal and antiviral components (e.g., double-stranded RNA, peptides, and chemicals) are discussed, which provides future prospects of VLP application in agricultural pest control. In addition, some concerns are raised about VLP production on a large scale and the short-term resistance of hosts to VLP uptake. Overall, this review is expected to stimulate interest and research exploring plant/insect virus-based VLP applications in agricultural pest management. © 2023 Society of Chemical Industry.     

一种快速简便检测马铃薯病毒的方法——病毒细菌协同凝集法(VBA)的研究

 在国内、外首次选用金黄色葡萄球菌(Staphylococc aureus)的No.180菌株用于病毒细菌协同凝集试验(Virobacterial agglutination test简称VBA)检测了来自马铃薯的4种不同形态粒子的病毒:马铃薯X病毒(PVX)、马铃薯丫病毒(PVY)、烟草花叶病毒(TMV)和马铃薯卷叶病毒(PLRV),其灵敏度达2.7~6.1ng/ml,检出病汁液的最大稀释度达10⁴~10⁵(PLRV1:500),较国内、外采用的Cown I菌株的灵敏度提高5~10倍。与血清学方法相比,VBA在2~3分种内就可获得结果,无假阳性反应,其灵敏度显著高于间接酶联法和免疫电镜,而接近于A蛋白酶联法。经抗血清致敏的菌体在4℃下保存4个月,对VBA检测的灵敏度没有影响。用VBA对采自田间的93个马铃薯病样进行检测,它们大多受2~3种病毒(PVX、PVY及PLRV)的复合侵染;和用间接酶联法及鉴别寄主检测的结果趋向一致。室内和田间试验均表明VBA灵敏度高、特异性强、快速简便和经济,尤其适合在基层单位中推广应用。