Purification and Serology of the Iranian Maize Mosaic Rhabdovirus1)

Leaves of maize infected with the Iranian maize mosaic rhabdovirus (IMMRV) were homogenized in 0.1 M citrate-0.04 M Na₂SO₃ buffer, pH 5.4, containing 10 % sucrose and the extract was subjected to low speed and high speed centrifugation followed by resuspension in 0.05 M potassium phosphate buffer, pH 7.2, containing 10 % sucrose. Partially purified preparation was obtained by density-gradient centrifugation, removal of the virus zones and their concentration by high speed centrifugation. Two virus specific bands were observed in density-gradient columns. An antiserum with a titer of 128 was prepared by injecting partially purified virus into rabbits. In agar-gel-diffusion tests, the antiserum produced one or two precipitin lines against diseased maize extract but none against healthy maize extract. IMMRV was not related to barley yellow striate mosaic (BYSMV), cereal chlorotic mottle (CCMV), Cynodon chlorotic streak (CCSV), Festuca leaf streak, and maize mosaic (MMV) viruses as well as to two unidentified rhabdoviruses occurring in wheat and Bermuda grass in the vicinity of Shiraz, when these viruses were tested against IMMRV antiserum in agar-gel-diffusion and enzyme-linked immunosorbent assay. Likewise, IMMRV did not react with antisera to BYSMV, CCMV, CCSV and MMV in agar-gel-diffusion tests. IMMRV appears to be different from most reported rhabdoviruses of cereals.     

Identification of a Strain of Peanut Chlorotic Streak Virus Causing Chlorotic Vein Banding Disease of Groundnut in India

A virus disease characterized by chlorotic vein banding, chlorotic line pattern along the margins or midrib of mature leaflets and chlorotic spots/rings was observed on commercial groundnut crops in Rayalaseema area of Andhra Pradesh with an incidence from 1% to nearly 60%. The virus was transmitted by mechanical inoculation in extracts prepared with 0.01 M potassium phosphate butter, pH 8.0 to 21 species from the Chenopodiaceae, Cruciferae, Leguminosae and Solanaceae, Chenopodium quinoa was found to be a good local lesion host. The virus was neither seed-transmitted through 1591 groundnut seeds nor aphid-transmitted by Aphis craccivora, Myzus persicae and Rhopalosiphum maidis either in non-persistent or semi-persistent manner. The virus remained infective in buffered tobacco leaf sap at a dilution of 10^?5; in a 10^?1 dilution of buffered sap the virus was infective for 2–3 days at 22–29°C or when heated to 65°C for 10 min but not to 70°C. Clarification treatments with organic solvents with 10% chloroform was least damaging. The virus was purified from Nicotiana rustica leaves. Purified virus contained isometric particles of 51 nm in diameter with an electron dense core of 22 nm and two major polypeptides of 76 kDa and 36 kDa. A polyclonal antiserum to this virus was produced. In agar gel double diffusion, enzyme-linked immunosorbent assay and in electro-blot immunoassay rests the virus was related to peanut chlorotic streak virus and not to cauliflower mosaic, figwort mosaic and soybean chlorotic mottle viruses.     

The etiology of hop chlorotic disease

Hop chlorotic disease was first described in England in 1930, but it has since been seldom seen and its etiology has remained unknown. In 1983 a patch of plants with the disease occurred in a large area of hops (Humulus lupulus) cv. Bramling Cross planted at Yalding, Kent in 1967. All plants in a rectangular area enclosing the disease outbreak were infected with hop mosaic, hop latent and prunus necrotic ringspot viruses; the diseased plants were additionally infected with arabis mosaic virus (AMV). The disease was also associated with seed-transmitted AMV, and was induced in hop seedlings inoculated with partially purified preparations of AMV originating from chlorotic disease-affected hops prepared from Chenopodium quinoa. The disease appears to be caused by AMV, but AMV isolates from hops with chlorotic disease were serologically indistinguishable from AMV isolates from hops with symptoms of bare-bine and/or nettlehead and showed similar pathogenicity in diagnostic hosts. The basis of the difference between isolates in their pathogenicity in hop remains unknown.     

杭州地区发生的玉米花叶病由甘蔗花叶病毒引起

从杭州地区呈现玉米矮花叶典型症状的玉米病组织中提纯得到大量线状病毒粒子,大多数长度为750?nm。病组织中含有大量风轮状内含体和板状集结体。病毒外壳蛋白为33.6 kD。病毒RNA13’端序列(1.8 kb)与甘蔗花叶病毒(SCMV)同源性最高,达71.5%～99.1%,与高梁花叶病毒(SrMV)同源性次之,为67.8%～68.5%,与玉米矮花叶病毒(MDMV)同源性最低,仅为38.4%～48.4%,从而初步认为此病害由SCMV引起。根据已发表的SCMV外壳蛋白氨基酸序列作亲缘性分析,表明SCMV可分为美国、南非、澳大利亚;德国和中国三大类。     

Purification and properties of arabis mosaic and tomato bushy stunt viruses isolated from lilac (Syringa vulgaris L.)

The paper gives more detailed characteristics of Arabis mosaic virus (AMV) and tomato bushy stunt virus (TBSV) isolated from lilac, the latter being identified in lilac (from plants suffering from yellow ring disease) for the first time. The isolate of TBSV from lilac, from which an antiserum with a titre of 1024 was prepared, is closely related to the artichoke strain. Information is given about two types of ringspot disease and about chlorotic ringspot of lilac. Whereas in the leaves of lilac suffering from ringspot disease (of ring mosaic type) the presence of AMV was demonstrated, the sap transmission from the leaves diseased with ringspot of linepattern (and wave-like mosaic) type failed; from the leaves affected by chlorotic ringspot a mixture of AMV and cherry leaf roll virus was identified. In addition, the polyetiological nature of “spring” mosaic and necrotic mosaic of lilac, in which bacteriumPseudomonas syringae van Hall, was found is dealt with. The TBSV was also identified in the isolate of necrotic mosaic.Additional index words: Lilac ringspot, chlorotic ringspot, yellow ring, “spring” mosaic, necrotic mosaic, cherry leaf roll virus,Pseudomonas syringae van Hall.     

Western Flower Thrips (Frankliniella occidentalis) Transmits Maize Chlorotic Mottle Virus

Maize chlorotic mottle virus (MCMV) is one of the co‐infection pathogens that cause corn (maize) lethal necrosis, but the transmission mechanism of MCMV is not yet clear. In order to determine the ability of western flower thrips (Frankliniella occidentalis; WFT) to transmit MCMV, imported maize seeds from Thailand were germinated in an insect‐free greenhouse and the seedlings were tested for the transmission by WFT of chlorotic mottle virus disease. The thrips (WFT), starved for 48 h then allowed to feed for 30 min on maize plants infected with MCMV or asymptomatic maize plants, were transferred to healthy seedlings. After 35 days, the seedlings with WFT from diseased maize plants showed chlorotic mottle symptoms, whereas seedlings with WFT from asymptomatic maize plants remained healthy. A single band of 711 bp was amplified by RT‐PCR using primers MCMV‐F/MCMV‐R from the MCMV‐infected plants and WFT collected from the diseased plants. Sequencing of the amplified product and further sequence comparison indicated that the two viruses from both sources showed 99% similarity of nucleotides and they should be regarded as identical. In addition, isometric particles c. 30 nm in diameter, characteristic of MCMV, were found in the WFT samples from diseased maize plants. Thus, it is concluded that WFT transmits MCMV. Our findings suggest that corn lethal necrosis disease can be controlled or minimized by the eradication of WFT from the field or greenhouses.     

Discrimination by gel-diffusion serology of digitaria striate, maize sterile stunt and other rhabdoviruses of Poaceae

Eight rhabdoviruses from grass and cereal hosts and their antisera were used to examine virus relationships by gel-diffusion serology. Nucleocapsid (Nc) preparations from digitaria striate virus (DSV) and maize sterile stunt virus (MSSV) both contained a major protein of c. 52 OOO daltons, and antisera prepared to these readily discriminated related planthopper-transmitted rhabdoviruses. MSSV showed a moderately close relationship to barley yellow striate mosaic virus (BYSMV) when an antiserum prepared to whole virus was used, but the Nc antiserum showed clearer discrimination. Worthern cereal mosaic virus and DSV showed a distant relationship to BYSMV and MSSV. There was no serological relationship between any of these viruses and cereal chlorotic mottle virus, cynodon chlorotic streak virus, festuca leaf streak virus or maize mosaic virus.     

Properties of some defective strains of tobacco mosaic virus and their behaviour as affected by inhibitors during storage in sap

From the type strain of tobacco mosaic virus, defective strains were isolated that produced chlorotic or ringspot type symptoms in tobacco and were difficult to transmit without carborundum in the inoculum. Their concentration was less than 0–1 μg/ml of sap instead of the usual 2 mg/ml with the type strain. Phenol extracts of infected leaves were a little more infective than extracts in buffer, whereas phenol extracts of leaves infected with type strain were very much less infective than extracts in buffer. Electron microscopy of infective sap rarely showed any virus particles, but preparations concentrated by ultracentrifugation contained virus particles, many of which were broken or seemed inadequately assembled. Changing the ambient temperature at which infected plants were kept from 20 to 35°C did not increase the amount or improve the appearance of the virus. Some of the strains were inactivated during heating for 10 min between 70 and 80 °C. Undiluted sap lost its infectivity in 3 days at 20 °C, as did the type strain when diluted to 0–1 μg/ml in sap from healthy leaves. This is because substances that inhibit infection were produced by microbes in the sap. The ability of sap from healthy leaves to inhibit infection increased by more than twenty-five times when left 3 days at 20 °C. Infectivity of appropriate mixtures of type strain and aged sap was restored by diluting them in buffer. Sodium azide at 0·02% in sap prevented formation of the inhibitor. The infectivity of the defective strains increased when inoculated together with the type strain.     

Isometric Virus-like particles in Abies alba Mill. and Other Abies Species: Partial Purification and Improved Detection by Means of Immunoelectron Microscopy

Abstract Two types of isometric virus-like particles (VLPs) with diameters of c. 20 and 35 nm were detected in partially purified extracts from Abies alba Mill., A. homolepis Sieb. et Zucc., and five other Abies species. An immunoelectron microscopical test was developed by means of which the large VLPs could often be detected even in crude sap preparations. They were to be widespread in A. alba and A. homolepis in the Federal Republic of Germany and Southern France. Theirpresence in glasshouse-grown seedlings of A. alba and A. homolepis shows that they are seed-transmissible. The small particles were detected only in partically purified preparations. Neither type of VLP was found in coniferous genera otherthan Abies.     

我国南方花生发生一种由蕃茄斑萎病毒引起的新病害

我们于1984和1985年6月上、中旬,在广州市郊、县,湛江市郊以及广西南宁市郊、县,北海市郊和合蒲县等花生产区,调查花生病毒病时,除了发现花生轻斑驳病毒病外,还发现一种新的病毒病害。其症状特征是:病株顶端叶片上出现很多褪绿黄斑或环斑,有的环斑变     

A Plant-Produced Pfs25 VLP Malaria Vaccine Candidate Induces Persistent Transmission Blocking Antibodies against Plasmodium falciparum in Immunized Mice

Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based ‘launch’ vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20–30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the ‘launch’ vector technology for the production of VLP-based recombinant vaccines against infectious diseases.     

Leaf mottling of Spartina species caused by a newly recognised virus, spartina mottle virus

The cause of a previously undocumented leaf mottling of Spartina species was investigated. Negatively stained preparations of sap from mottled leaves revealed flexuous particles 725 × 12 nm. Pinwheels with associated laminar inclusion bodies were observed in thin sections of affected mesophyll cells. The virus was purified from infected Spartina anglica plants and had a sedimentation coefficient in 0·015 m borate of 150S. The virus was transmitted by inoculation of sap to healthy Spartina anglica, but not to a range of other graminaceous or dicotyledonous species tested. It was distantly serologically related to agropyron mosaic virus, but not to other viruses with similar morphology; the name spartina mottle virus is proposed.     

The occurrence of maize mosaic virus on sorghum in India1

A leaf disease of sorghum (Sorghum bicolor) characterised by fine discontinuous chlorotic streaks between the veins, was observed on sorghum grown during the 1987/88 post-rainy season in peninsular India. Early-infected plants were stunted, had shortened internodes, and produced poorly developed panicles. The virus was transmitted by the delphacid planthopper, Peregrinus maidis. Negatively stained leaf dip preparations contained bullet-shaped virus particles (208 ± 4.4 × 66 ± 1.0 nm) resembling those of rhabdoviruses. In ultrathin sections, the particles budded through the inner nuclear membrane and were present in the cytoplasm within membrane-bound vesicles that were apparently contiguous with the distended outer nuclear membrane. A method for purifying the virus was developed utilising polyethylene glycol (PEG) precipitation, Celite filtration and sucrose densitygradient centrifugation. An antiserum was produced in rabbits with a titre of 1/2650 in the precipitin ring interphase test. The virus could be detected in infected sorghum leaf tissues using a direct antigen coating form of enzyme-linked immunosorbent assay (DAC-ELISA). In immuno-double diffusion tests, the virus reacted positively with antisera to maize mosaic virus (MMV) from Reunion (MMV-RN) and Hawaii (MMV-HI), but not with antisera to barley yellow striate mosaic (BYSMV), cereal chlorotic mottle (CCMV), and cynodon chlorotic streak (CCSV) viruses. Thus, the virus isolated from sorghum is designated the MMV-S isolate. In DAC-ELISA tests, MMV-S reacted positively with antisera to MMV-R, MMV-HI, MMV-Florida isolate, CCSV, and CCMV, and weakly with antiserum to BYSMV. SDS-polyacrylamide gel electrophoresis revealed four major proteins of relative mass M_r 70 000, 59 000, 32 000 and 28 000. In electro-blot immunoassay, MMV and CCSV antisera detected the G and N proteins. These data suggest that MMV-S should be placed in the sonchus yellow net virus subgroup of plant rhabdoviruses.     

Purification and Partial Characterization of Isometric Virus-like Particles in Kalanchoe Species

Isometric virus-like particles (IVLP) were detected in crude sap from Kalanchoe pinnata, K. daigremontiana and K. tubiflora plants showing a mild mosaic on the leaves. These particles of 35 nm in diameter were transmitted mechanically to several test plants but not to healthy Kalanchoe. Air temperatures above 30 °C hindered the infection process. The IVLP were purified from systematically infected Nicotiana benthamiana using Triton X-100 as clarifying agent followed byP, EG precipitation. IVLP were degraded by organic solvents and formed aggregates in the presence of 2 mmol/1 CaCl₂. The particles occurred in relatively low concentration in plant sap and lost infectivity in leaves frozen at -70 °C for one week and in purified preparations kept at 4 °C. In buffer crude sap of N. benthamiana IVLP had a thermal inactivation point between 45 and 50 °C on a longevity in vitro of 20 h at 25 °C. Particles contained one nucleoprotein component witha molecular weight of 46,000 daltons and a ssRNA species which, when denatured, had a molecular weight of 1.2 × 10⁶. IVLP purified preparations exhibited a typical nucleoprotein absorption spectrum with a maximum at 254–260 nm and a minimum at 240,–243 nm and a A 260/280 ratio of 1.56. The buoyant density of the IVLP was 1.32 g/ml calculated by isopycnic centrifugation on CsCl. Ultrastructural studies in infected leaves of K. pinnata indicated that IVLP caused an increase in chloroplast volume, distortion of the grana and reduced the number of thylakiods per grana. IVLP infection also impared the diurnal pattern of synthesis and hydrolysis of starch, characteristic of CAM plants. The non-serological reaction of the IVLP with antisera specific to members of 7 different groups of spherical viruses as well as the combination of physicochemical properties and host range, exhibited by these particles impeded their taxonomic location. In nature, young Kalanchoe plantlets acquire the IVLP through their physical connections with the infected mature leaves.     

Biological factors affecting leafhopper transmission of purified maize chlorotic dwarf machlovirus

A helper component from maize chlorotic dwarf machlovirus (MCDV)-infected plants was necessary for insect transmission of purified MCDV. Purified MCDV, WS strain, when acquired by membrane feeding, was transmitted byGraminella nigrifrons (Forbes) (Homoptera: Cicadellidae) which fed first on MCDV (M1 strain)-infected maize. Conversely, feeding on MCDV-WS-infected maize allowed transmission of purified MCDV-M1, indicating that the helper component was not strain specific. ViruliferousG. nigrifrons lost the ability to transmit MCDV-M1 after feeding for 24 h on healthy maize, but retained the ability to acquire and transmit purified MCDV-WS for up to 36 h. Wheng. nigrifrons fed initially on MCDV-infected plants and then on a second strain of purified MCDV, the second strain of MCDV could be transmitted alone, suggesting that the helper component from MCDV-infected plants was something other than the virion itself. Lengthening acquisition feeding ofG. nigrifrons on MCDV-M1-infected maize or purified MCDV-WS did not significantly change the transmission frequency of MCDV-WS.Amblysellus grex (Oman), an experimental vector of MCDV, also transmitted purified MCDV-WS after an initial acquisition feeding on MCDV-M1-infected maize. This suggests that the helper component is not vector species specific.     

Apple mosaic virus isolated from hop plants in Japan

A strain of apple mosaic virus was isolated from hop plants in Japan. The virus was purified from young hop plants and back-inoculated to virus-free hop plants obtained by meristem tip culture. Inoculated plants developed chlorotic spots, ringspots and a band pattern accompanied by necrosis in the inoculated and systemically infected leaves. Shoot tips of infected plants sometimes became necrotic and these symptoms resembled those of a ring- and band-pattern mosaiclike disease prevalent in hop gardens in Japan. Since apple mosaic virus was recovered from infected plants, it is likely that the virus was the causal agent of this disease. Agar gel double diffusion tests and ELISA showed the hop virus to be serologically closely related to apple mosaic virus (ApMV), and distantly related to prunus necrotic ringspot virus (PNRSV). The virus had a narrow host range, and infected only cucumber of 18 species of Cucurbitaceae, Chenopodiaceae, Leguminosae or Solanaceae inoculated. It produced chlorotic spots on the inoculated cotyledons of cucumber, but no systemic infection. By contrast, ApMV from apple and PNRSV from peach had wide host ranges and infected cucumber plants systemically.     

A yellow mosaic disease of horse chestnut (Aesculus spp.) caused by apple mosaic virus

A severe foliar yellow mosaic disease was observed in horse chestnut trees (Aesculus carnea and A. hippocastanum). Reactions in woody indicator plants grafted with diseased horse chestnut suggested the presence of an ilarvirus. Virus isolates obtained by mechanical inoculation of herbaceous test plants reacted with antisera to apple mosaic virus but not with antisera to its serotype prunus necrotic ringspot virus, or to prune dwarf virus. Yellow mosaic was induced in horse chestnut seedlings grafted with tissues from herbaceous hosts infected with horse chestnut isolates or with the European plum line pattern isolate of apple mosaic virus. Virus was detected by enzyme-linked immunosorbent assay (ELISA) in embryo and endosperm of immature seed from infected trees but not in mature seed, or progeny seedlings. Strawberry latent ringspot virus was detected in one of six A. hippocastanum trees with a leaf vein yellows disease.     

Influenza virus‐like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice

A strain‐specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant‐based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high‐molecular‐weight structures. H5‐containing structures were purified and examination by transmission electron microscopy confirmed virus‐like particle (VLP) assembly. High‐performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP‐producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 µg of purified influenza H5‐VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 µg of H5‐VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.     

Involvement of Volatile Substances in Systemic Resistance of Barley Against Erysiphe graminis f. sp. hordei Induced by Pruning of Leaves

Horsegram yellow mosaic disease was shown to be caused by a geminivirus; horsegram yellow mosaic virus (HYMV). The virus could not be transmitted by mechanical sap inoculation. Leaf dip and purified virus preparations showed geminate virus particles, measuring 15-18 * 30 nm. An antiserum for HYMV was produced and in enzyme-linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) tests HYMV was detected in leaf extracts of fieldinfected bambara groundnut, french bean, groundnut, limabean, mungbean, pigeonpea and soybean showing yellow mosaic symptoms. Bemisia tabaci fed on purified HYMV through a parafilm membrane transmitted the virus to all the hosts listed above but not to Ageratum conyzoides, okra, cassava, cowpea, Croton bonplandianus, Lab-lab purpureus, Malvastrum coromandalianum and tomato. No reaction was obtained in ELISA and ISEM tests between HYMV antibodies and extracts of plants diseased by whitefly-transmitted agents in India such as A. conyzoides yellow mosaic, okra yellow vein mosaic, C. bonplandianus, yellow vein mosaic, M. coromandalianum yellow vein mosaic, tomato leaf curl and cassava mosaic. HYMV was also not found to be related serologically to bean golden mosaic, virus.     

THE INFLUENCE OF LIGHT INTENSITY ON THE SUSCEPTIBILITY OF PLANTS TO CERTAIN VIRUSES

Reducing the light intensity under which plants were grown in summer to one-third increased their susceptibility to infection with tobacco necrosis, tomato bushy stunt, tobacco mosaic and tomato aucuba mosaic viruses. With the first two viruses shading increased the average number of local lesions per leaf by more than ten times and by more than five times with the second two.
Reducing the light intensity increased the virus content of sap from leaves inoculated with Rothamsted tobacco necrosis virus by as much as twenty times. As it also reduced the total solid content of sap by about one-half, purification was greatly facilitated; crystalline preparations of the virus were readily made from shaded plants but not from unshaded controls.
Reducing the light intensity also increased the virus content of systemically infected leaves; the greatest effect was with tomato bushy stunt virus with which increases of up to ten times were obtained, but with tobacco mosaic and aucuba mosaic viruses there were also significant increases.
The importance of controlled illumination in raising plants for virus work and the possible mechanisms responsible for the variations in susceptibility are discussed.