20. Electro-Extraction of Viruses from Infected Plant Tissue (Applied to Turnip Yellow Mosaic,Tobacco Mosaic,and Maize Streak Viruses)

ABSTRACT

An apparatus is described which was used for rapid extraction of viruses from frozen and thawed infected plant tissues. The novel principle is the establishment of a potential gradient of 15 to 20 volts/cm at approximately 90° across the leaves are surrounded by buffer of low molarity and of the appropriate hydrogen ion concentration. To keep the leaves in the correct orientation they were placed as single layers between coarse rigid plastic gauze. The method, termed electro-extraction, was used as the initial step in the purification of turnip yellow mosaic, tobacco mosaic and maize streak viruses. An electron micrograph of the purified maize streak virus is presented.     

Peanut stripe virus - a new seed-borne potyvirus from China infecting groundnut (Arachis hypogaea)1

A new virus, peanut stripe (PStV), isolated from groundnut (Arachis hypogaea) in the USA, induced characteristic striping, discontinuous vein banding along the lateral veins, and oakleaf mosaic in groundnut. The virus was also isolated from germplasm lines introduced from the People's Republic of China. PStV was transmitted by inoculation of sap to nine species of the Chenopodiaceae, Leguminosae, and Solanaceae; Chenopodium amaranticolor was a good local lesion host. PStV was also transmitted by Aphis craccivora in a non-persistent manner and through seed of groundnut up to 37%. The virus remained infective in buffered plant extracts after diluting to 10^-3, storage for 3 days at 20°C, and heating for 10 min at 60°C but not 65°C. Purified virus preparations contained flexuous filamentous particles c. 752 nm long, which contained a major polypeptide of 33 500 daltons and one nucleic acid species of 3·1 × 10⁶ daltons. In ELISA, PStV was serologically related to blackeye cowpea mosaic, soybean mosaic, clover yellow vein, and pepper veinal mottle viruses but not to peanut mottle, potato Y, tobacco etch, and peanut green mosaic viruses. On the basis of these properties PStV is identified as a new potyvirus in groundnut.     

Isolation of A Cytoplasmic Polyhedrosis Virus by Physical and Immunological Techniques

Procedures are described for the separation and purification of cytoplasmic polyhedrosis viruses obtained from multiply infected Heliothis armigera larvae. Separation was achieved by differential centrifugation and density gradient zone electrophoresis followed by complexing with nuclear polyhedrosis virus specific antibody. The yield of cytoplasmic polyhedrosis virus was increased by passage in larvae reared on synthetic media.     

Polypeptide composition of the purified Photosystem II pigment-protein complex from spinach

The Photosystem II pigment-protein complex, the chlorophyll α-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine.The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43 000 and 27 000. The chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100°C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43 000 and 27 000, are valid structural or functional components of Photosystem II pigment-protein complex.     

马铃薯Y病毒组病毒高产量提取方法的建立

本文报道了高产量提取芜菁花叶病毒（ＴｕＭＶ）、莴苣花叶病毒（ＬＭＶ）、芋花叶病毒（ＤＭＶ）和大豆花叶病毒（ＳＭＶ）的提取方法。本方法通过使用高盐浓度的磷酸盐缓冲液以及在缓冲液中加入氯化镁和脲,并用ＴｒｉｔｏｎＸ－１００作为澄清剂,替代常规使用的氯仿和正丁醇,成功的提取到了大量病毒粒子,上述四种病毒提取的得率分别是ＴｕＭＶ为１７３．３ｍｇ／ｋｇ病叶,ＬＭＶ为９６ｍｇ／ｋｇ病叶,ＳＭＶ为１９９．２ｍｇ／ｋｇ病叶,ＤＭＶ为１７６．６ｍｇ／ｋｇ病叶。     

18. Isolation of a Cytoplasmic Polyhedrosis Virus by Physical and Immunological Techniques

ABSTRACT

Procedures are described for the separation and purification of cytoplasmic polyhedrosis viruses obtained from multiply infected Heliothis armigera larvae. Separation was achieved by differential centrifugation and density gradient zone electrophoresis followed by complexing with nuclear polyhedrosis virus specific antibody. The yield of cytoplasmic polyhedrosis virus was increased by passage in larvae reared on synthetic media.     

High temperature inactivation of cucumber and alfalfa mosaic viruses in Nicotiana rustica cultures

Cucumber mosaic (CMV) and alfalfa mosaic (AlfMV) viruses could not be detected in Nicotiana rustica tissues cultured at 32 °C for 16–18 days or at 40 °C for 5 days, but infectivity remained high in comparable tissue cultured at 22 °C. Incubation of infected cultures at 28–30 °C resulted in an initial reduction followed by a partial recovery in the infectivity of both viruses. The infectivity of CMV in tissues grown between 12 and 32 °C was highest in cultures grown at 12 °C. Although CMV infectivity was not detected in cultures after 16–18 days at 32 °C, virus was eliminated only after a further 30 days at 32 °C. When cultures were transferred from 32 to 22 °C after shorter treatment periods, infectivity rapidly increased to levels higher than those of infected tissues grown continuously at 22 °C. At 40 °C, CMV was eliminated from infected tissues after 9 days and AlfMV after 7 days. Cultures grown continuously at 40 °C deteriorated rapidly but, when grown under diurnal alternating periods of 8 h at 40 °C and 16 h at 22 °C, they remained viable and CMV was also inactivated.     

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica)

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.     

Eggplant mosaic virus, and its relationship to Andean potato latent virus

Eggplant mosaic virus (EMV), obtained from Solanum melongena L. from Trinidad, is readily transmitted by inoculation of sap to several solanaceous and a few non-solanaceous plant species. Purified preparations of EMV contain isometric particles 30 nm in diameter, and with sedimentation co efficients of either 111 or 53 S. The particles have thirty-two major morphological subunits. EMV is closely serologically related to Andean potato latent virus and has a similar host range, but is more virulent. Also, whereas EMV accumulates fastest in Nicotiana clevelandii leaves at 20–24 °C, Andean potato latent virus accumulates fastest at 15 °C, and fails to attain a serologically detectable concentration at 24 °C. A few symptomatologically or serologically distinguishable strains of EMV were obtained. EMV has properties typical of viruses of the Andean potato latent subgroup of the turnip yellow mosaic group of viruses, and its present cryptogram is */*:*/*:S/S:S/Cl.     

Identification, purification and some properties of a mosaic virus of okra (Hibiscus esculentus)

In the Ivory Coast, an apparently undescribed virus was isolated from okra (Hibiscus esculentus) in which it caused mosaic and leaf vein banding. The virus was sap transmissible to a wide range of plants and had a thermal inactivation point of 80 °C. It was named okra mosaic virus (OMV). A purification procedure was developed. Electron microscopy and analytical and density gradient centrifugation showed that OMV was an isometric virus accompanied by empty shells (top component). Serological tests showed OMV to be a member of the tymovirus group.     

Auswirkungen des Anbaus einer Wirts- und einer Nichtwirtspflanze unter wechselnden ökologischen Bedingungen auf die Populationsdynamik von Rhizoctonia solani Kühn und Fusarium solani f. pisi (Jones) Snyd. et Hans

An isometric virus was isolated from Helianthus annuus L. plants showing a yellow leaf spot mosaic on affected leaves. Infected plants were found in different ecological regions of Ukraine. A procedure of virus purification is described. The diametres of the virus particles were nonuniform and ranged from 50 to 120 nm. The sedimentation coefficient of the virus was 518–540 S and the floating density in the CsCl gradient was 1.22 g/cm³. The MW of proteins separated by electrophoresis amounted to 78±0.9, 58±0.8, 52±0.2, and 27±0.8 kDa, respectively. The virus was assigned to the tospoviruses for which sunflower is a new previously undescribed natural host plant.     

侵染半夏的两种病毒的分离纯化和初步鉴定

用自然感染的半夏（Ｐｉｎｅｌｌｉａｔｅｒｎａｔａ）为材料,经粗提纯后检查到一种线状病毒和一种球状病毒,用两种方法对担提纯样品中的病毒粒子进行了分离纯化。１０％－７０％连续甘油梯度８００００ｇ离心１５０分钟获得两条病毒带,经紫外吸收测定均为强的核蛋白吸收峰,病毒粒子检查分别为球状和线状病毒粒子,线状病毒经浓缩收集为均一成份．与芋花叶病毒（Ｄａｓｈｅｅｍｍｏｓａｉｃｖｉｒｕｓ,ＤＭＶ）抗体有强的阳性反应。粗提纯样品经０．８％琼脂糖凝胶电脉分离为一条蛋白带,该条带回收后经紫外吸收测定为核蛋白吸收峰,电镜下检查为均一的球状病毒,以ＴＭＶ为对照、醋酸铀（ＵＡＣ）负染后测得该球状病毒（ｐｉｎｅｌｌｉａｓｐｈｅｒｉｃａｌｖｉｒｕｓ１．ＰｓＶ－１）的大小为３１．７ｎｍ;戊二醛固定后磷钨酸（ＰＴＡ）负染测得ＰｓＶ—１的大小为３４．０ｎｍ。各组分经ＳＤＳ－聚丙烯酸胺凝焦电泳分析测得线状病毒和球状病毒的外壳蛋白分子量分别为２０ＫＤ和２８ＫＤ。初步确定线状病毒为ＤＭＶ．球状病毒ＰｓＶ—１为侵梁天南星科半夏的一种新病毒。     

Some hosts and properties of dahlia mosaic virus

Dahlia mosaic virus (DMV) infected twenty-five of the eighty-five plant species from four of eighteen families inoculated, but only dahlias were found naturally infected. DMV infected fourteen members of the Solanaceae, Amaranthaceae and Chenopodiaceae, and eleven of twenty-nine Compositae. Verbesina encelioides was the best plant for diagnosis, assay and source of virus. Systemically infected hosts contained ovoid intracellular inclusions 2–5–10 μm in diameter which were shown by electron microscopy to consist of a finely granular, vacuolated matrix containing numerous virus particles. V. encelioides sap was sometimes infective after dilution to 1/2000 but not 1/3000, after heating for 10 min to 75 °C but not 80 °C, and after 4 days at 18 °C or 32 days at 2 °C. Sap from infected dahlia, Zinnia elegans or Ageratum houstonianum rapidly became non-infective, but extracts made with 0·05 M sodium thioglycollate or 0·03 M sodium diethyldithiocarbamate remained infective for 24–48 h at 18 °C. Some purified preparations remained infective for up to 3 years at 2 °C. DMV was best purified from V. encelioides by one or more cycles of differential centrifugation, followed by density-gradient centrifugation and further concentration. Composition, molarity, and pH of the extracting buffer had little effect on yield of virus. Best yields were obtained from extracts stored with 8-5% (v/v) n-butanol at 2 °C for 10–14 days. Purified preparations were infective at dilutions up to 1/5000, had ultraviolet absorption spectra typical of a nucleoprotein (Å 260/280 = 1·47), probably contained DNA, and had a single sedimenting component having isometric particles c. 50 nm in diameter with a sedimentation coefficient of 254 S. The cryptogram of DMV is (D)/*:*/(16):S/S:S/Ap. DMV is serologically closely related to cauliflower mosaic virus, but the viruses are distinct pathogens. The two viruses have similar properties, size, shape and other characteristics, and together with at least three others form a small but apparently homogeneous group of aphid-borne viruses.     

Purification and some properties of an extracellular ribonuclease with antiviral activity against tobacco mosaic virus from <Emphasis Type="Italic">Bacillus cereus</Emphasis>

A new ribonuclease (RNase) with tobacco mosaic virus inhibition was isolated and purified from Bacillus cereus ZH14 through ammonium sulfate precipitation, ultrafiltration, ion-exchange chromatography of DEAE-Sephadex A-50 column, and gel chromatography of Sephacryl S-200HR column. The enzyme was purified approximately 134-fold with a recovery of 9.2%. The RNase had an MW of 75.6 kDa in SDS-PAGE, which differed from RNases reported previously. The inhibitory activity of the RNase in the purification process against tobacco mosaic virus was tested, and the percentage inhibition of the purified RNase (48 U/ml) reached 90%. The protein could tolerate 90°C and pH 4.0.     

Isolation of viral double-stranded RNAs using a LiCl fractionation procedure

A general procedure for the isolation of virus-specific double-stranded RNA (ds-RNA) is discribed. The procedure is based on the differential solubility of different types of nucleic acids in LiCl. Principal advantages over conventional methods are simplicity, avoidance of enzymatic treatment, and relatively good yields of undegraded ds-RNA while permitting separation of several main groups of cellular and viral nucleic acids from the same batch of tissue. The method has been successfully applied in tissues infected by several representative plant RNA viruses. The virus-specific ds-RNAs obtained have been identified by their resistance to ribonuclease and comparison of their electrophoretic mobilities with those of the corresponding single-stranded RNA (ss-RNA) in polyacrylamide gels. The molecular weights of the ds-RNAs of tobacco mosaic virus, turnip yellow mosaic virus, alfalfa mosaic virus, and peanut stunt virus fit the curved log molecular weight-migration relationship constructed from a set of known marker ds-RNAs.     

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.     

Formaldehyde-induced tight binding of protein to RNA in particles of rod-like virus

The formaldehyde-induced formation of tightly bound RNA-protein complexes of rod-like plant viruses was studied. The preparations of tobacco mosaic virus and closely related cucumber virus 4 were incubated with 1.5% formaldehyde for 20-50 hrs at 50 degrees C. Then the viral particles were disrupted, free protein was removed and viral RNA was centrifuged in the linear gradient of Cs2SO4. The RNAs from the formaldehyde-untreated viruses and RNA from the formaldehyde-treated tobacco masaic virus had the density of 1.65-1.66 g/cm3, while RNA from the formaldehyde-treated cucumber virus had the density of 1.57-1.42 g/cm3, depending on the incubation time. This is indicative of the protein binding to RNA. Treatment of the cucumber virus complex with pronase resulted in a liberation of free RNA with the density of 1.66 g/cm3; incubation for 2 min at 100 degrees C in a dissociating mixture (2% sodium dodecyl sulfate + 0.2% mercaptoethanol) did not cause the dissociation of the complex. Polyacrylamide gel electrophoresis showed that the most part of the protein molecules are bound within the complex not by covalent protein-protein cross-links.     

Peanut Chlorotic Ringspot Virus (PCRV), a Newly Discovered Virus Infecting Peanut (Arachis hypogaea L.)

A virus causing wide chlorotic ringspot (PCRV) associated with chlorotic line pattern and motthng on an Erictoides hybrid growing in USDA-OSU greenhouses, Stillwater, Oklahoma, was discovered. The virus was isolated and characterized and found to differ in symptomology, host range and serological properties from all the previously described viruses infecting peanut, particularly those reported in the United States to be the most important ones, peanut mottle virus, peanut stripe virus, and tomato spotted wilt virus. The virus was transmitted by both mechanical inoctilation and grafting to fourteen peanut cultivars causing identical symptoms to those originally observed on the Erictoides hybrid. In addition to peanut, the virus systemically infected Pisum sativum L. ‘Little marvel’ causing mainly mosaic and Lupinus albus L. ‘Tiftwhite’ producing severe malformation and remarkable reduction in leaflet area. The virus did not infect many other plant species of which cowpea ‘California blackeye’ (Vigna unguiculata L.) and at least five cultivars of soybean (Glydne max L.) are known to be susceptible hosts to peanut mottle virus. Phaseolus vulgaris L. ‘Topcrop’ and Chenopodium amaranticohr Coste & Reyn were found to be two useful local lesion assay and diagnostic hosts for PCRV. The virus elicited necrotic local lesions on the first and chlorotic ringspots on the second. PCRV had a dilution end point between 10^?5 and 10^?6, thermal inactivation point between 55°C and 60°C, and longevity in vitro up to 6 days but not 7 days. Virus particles viewed hy electron microscopy and the negative stain uranyl aceute were flexuous filamentous particles ranging in length from 750–850 nm. In both indiren PAS-ELISA and Ouchterlony double immunodiffusion test, PCRV was serologically related to a PMV isolate from Oklahoma (PMV-OK.) but not to bean yellow mosaic virus, peanut stripe virus, potato virus Y, watermelon mosaic virus 1, watermelon mosaic virus 2, wheat streak mosaic virus, and zucchini yellow mosaic virus.     

Some hosts, properties and possible affinities of a labile virus from Hypochoeris radicata (Compositae)

Hypochoeris mosaic virus (HMV) is common in Hypochoeris radicata (‘cat's ear’) in western Canada. It infected 10 of 53 mechanically inoculated species in five of twelve families, but was not transmitted by aphids or through seed or soil. Sap from infected Nicotiana clevelandii was sometimes infective after dilution to 10^-1 and occasionally 10², after 10 min at 45 but not 50°C, and after 1 but not 2 days at 20°C. Infectivity of crude nucleic acid extracts from infected leaves was rapidly abolished by RNase but not by DNase. Host sap contained very few rod-shaped particles or particle fragments mostly 21.0–22.5 nm in diameter, and up to 420 nm long but with predominant lengths of 120–140 and 240–260 nm. Many rods in purified virus preparations were less than 240 nm long, and the majority were c. 140 nm or shorter. The particles had a helical substructure with a pitch of 2.58 nm and contained a single type of protein of estimated mol. wt 24.5 × 10³. HMV showed no serological relationship to eight morphologically similar viruses (beet necrotic yellow vein, broad bean necrosis, barley stripe mosaic, peanut clump, potato mop-top, Nicotiana velutina mosaic, wheat soil-borne mosaic and defective strains of tobacco mosaic). It is probably a hitherto undescribed tobamovirus.     

East African strains of cowpea aphid-borne mosaic virus

Cowpea aphid-borne mosaic virus (CAMV) was isolated for the first time in East Africa where three distinct strains, type, veinbanding and mild, were differentiated by host range and serology. The three strains infected 17/38, 18/37 and 10/35 legume species, and 11/21, 7/21 and 3/19 non-legume species, respectively. The viruses were propagated in cowpea and assayed in Chenopodium amaranticolor. Isolates of all three strains had similar in vitro properties: dilution end point between 10^-3 and 10^-4; thermal inactivation point between 56 and 58 °C; longevity in vitro between 2 and 3 days. Infectivity of sap from frozen leaves was high after 4 wk but much less after 7 wk; infectivity was largely precipitated by 50% acetone but inactivated by 50% ethanol. High yields of virus were consistently obtained from cowpea by extracting systemically infected leaves in 0.5 m sodium citrate containing 1% mercaptoethanol (pH 8.1), and clarifying with 8.5 ml n-butanol/100 ml sap. Virus preparations contained numerous unaggregated and aggregated virus particles c. 750 nm long and contained components with sedimentation coefficients (s°_{20, w}) of 150S and 175S (presumably unaggregated and aggregated particles, respectively). CAMV is serologically distantly related to bean common mosaic virus, but not to bean yellow mosaic or eight other morphologically similar viruses. It is a typical but distinct member of the potato virus Y group.