Rod-shaped Virus Particles Research Articles

Construction of Full-Length cDNA Clones to Soil-Borne Wheat Mosaic Virus RNA1 and RNA2, from Which Infectious RNAs Are Transcribed In Vitro: Virion Formation and Systemic Infection without Expression of the N-Terminal and C-Terminal Extensions to the Capsid Protein

The 19-kDa capsid protein (CP) of Soil-borne wheat mosaic furovirus (SBWMV) is encoded in the 5′-terminal region of RNA2. In addition to CP, two CP-related proteins are translated from SBWMV RNA2: (1) a 24-kDa protein (N-CP) with an N-terminal 40-amino-acid extension initiated at an upstream in-frame CUG codon; and (2) an 83-kDa protein (CP-RT) with an about 580-amino-acid, C-terminal extension by partial translational readthrough at the UGA termination codon at the end of the CP gene. We examined requirements for N-CP and CP-RT on virion formation and systemic infection in wheat plants using full-length cDNA clones, from which infectious RNA can be transcribed in vitro. RNA2 mutants, which could not synthesize N-CP, CP-RT, or either infected wheat plants systemically in combination with the wild-type RNA1 transcripts, produced rod-shaped virus particles in uninoculated upper leaves. Original mutations which abolished translation of N-CP and CP-RT were confirmed on RNA2 extracted from purified virus from the upper leaves by nucleotide sequence analysis. These results indicate that neither N-terminal nor C-terminal extensions to the CP are required for virion formation and systemic infection of SBWMV in wheat plants.

NMR Measurement of Dipolar Couplings in Proteins Aligned by Transient Binding to Purple Membrane Fragments

One-bond internuclear dipolar couplings in molecules that are aligned relative to an external magnetic field constrain the orientations of the corresponding vectors relative to the molecule’s alignment frame.1-3 Such couplings can be measured easily if the molecular alignment is very weak, such that only the large one-bond effects are observed as a change in the corresponding one-bond J coupling, and broadening resulting from more remote interactions is negligible. For biological macromolecules such as proteins and nucleic acids, the required small degree of alignment with the magnetic field sometimes can be obtained as a result of their own magnetic susceptibility anisotropy,2,3 or more generally, it can be induced by dissolving them in an anisotropic medium.1 Lyotropic liquid crystalline media, consisting of planar phospholipid particles known as bicelles,4 are particularly useful for this purpose.5 Very recently, it has been demonstrated that nematic phases of rod-shaped virus particles can also be used for obtaining a tunable degree of solute alignment.6,7 In both cases solute alignment is contingent upon arrangement of individual particles (bicelles or viruses) in a liquid crystalline phase, which collapses below a given threshold concentration. Therefore, there is a lower limit for the degree of solute alignment which can be obtained in such media. Also, there are a number of proteins which destructively interfere with the bicelle liquid crystalline phase and for which dipolar couplings in such media cannot be measured.5,6 Similarly, it is likely that certain systems will prove unsuitable for measurement in the virus-based media. If a macromolecule can be studied in two or more different oriented media, which in general yield different macromolecular alignment tensors, this dramatically increases the structural information contained in the dipolar couplings.8 Here, we demonstrate that a suspension of planar purple membrane (PM) fragments, containing bacteriorhodopsin (BR), can be used to yield the required weak degree of macromolecular alignment in a strong magnetic field. The magnetic susceptibility anisotropy of these PM fragments is dominated by the membrane spanning helices of BR. Their large size (diameter of 0.2 to 2 μm)9 and high BR content (75%) result in essentially full alignment of individual particles at field strengths g10 T.9 Unlike the liquid crystalline case, there is no critical lower threshold for their concentration. The PM fragments are highly negatively charged, and the average alignment of two water-soluble proteins is shown to be dominated by electrostatic interactions. The solute alignment tensor obtained in the PM medium, therefore, is expected to be quite different from that in virusor bicelle-based liquid crystals. Measurement of one-bond dipolar couplings is demonstrated for two 15N-labeled proteins, the VR domain of the human T-cell receptor, which is one of the proteins which could not be studied in the bicelle medium,5 and ubiquitin which has been studied extensively by NMR and crystallography.10,11 Figure 1 shows a small region of the IPAP 1H-15N HSQC spectrum,12 recorded for 0.4 mM of the VR domain, in a suspension containing 3 mg/ mL purple membrane fragments, 100 mM NaCl, pH 7.1. Substantial deviations from the isotropic JNH splitting (ca. 94 Hz) can be seen, indicating significant alignment. In the frame of the diagonalized molecular alignment tensor, the dipolar couplings can be described by the equation

First Report of Cassava Common Mosaic Potexvirus Infecting Chaya (Cnidoscolus chayamansa) in Tuvalu.

Chaya (Cnidoscolus chayamansa McVaugh) was introduced into Tu-valu as a leaf vegetable in the early 1990s (1) with the primary objective of alleviating vitamin A and C deficiencies, which at that time were prevalent throughout the population. Although there are no definitive quarantine records, we believe that the crop originated from vegetative cuttings introduced into Funafuti atoll from Kiribati. In the years following its introduction, chaya was distributed as cuttings taken from the original, introduced germplasm, to other islands and atolls of Tuvalu. The occurrence of yellow patches on the mature leaves of chaya cultivated in Funafuti had previously been attributed to iron deficiency, which was the inevitable consequence of growing the crop on coral atoll soils that are well known to be poor sources of metallic ions, notably iron, zinc, and copper. Following heavy cyclonic rains in March 1997, young, emerging chaya leaves were noted to exhibit an angular mosaic of the leaf lamina, characteristic of virus infections. Negatively stained sap from mosaic-affected leaves, when examined in the transmission electron microscope, was found to contain many rod-shaped virus particles similar in morphology to those of the Potexviridae (approximately 510 × 15 nm). The virus was transmitted mechanically to Chenopodium amaranticolor, in which it induced local lesion symptoms. The virus was found to be more closely related serologically to cassava common mosaic potexvirus (CsCMV) from Brazil than to that from Colombia, by immunosorbent electron microscopy with antisera kindly provided by F. Morales. Recently, CsCMV particles have been found in mosaic-affected chaya derived from the original Funafuti mother plants on Vaitupu Island. Similar symptoms have been observed on chaya being cultivated in Nui and Nanumaga islands. We conclude that CsCMV must now have been distributed throughout the atolls and islands of Tuvalu by infected planting material. We believe this to be the first report of the occurrence of CsCMV outside Central America and the first record of a plant virus from Tuvalu. Reference: (1) C. Howells and M. Bainbridge. 1996. South Pacific Commission, Pacific Island Forests and Trees. Page 10.

Characterization of petunia flower mottle virus (PetFMV), a new potyvirus infecting Petunia x hybrida.

With the introduction of cutting-grown Petunia x hybrida plants on the European market, a new potyvirus which showed no serological reaction with antisera against any other potyviruses infecting petunias was discovered. Infected leaves contained flexuous rod-shaped virus particles of 750-800 nm in length and inclusion bodies (pinwheel structures) typical for potyviruses in ultrathin leaf sections. The purified coat protein with a Mr of approximately 36 kDa could be detected in Western immunoblots with a specific antibody to the coat protein of the petunia-infecting virus. The 3' end of the viral genome encompassing the 3' non-coding region, the coat protein gene, and part of the NIb gene was amplified from infected leaf material by IC/PCR using degenerate and specific primers. Sequences of PCR-generated cDNA clones were compared to other known sequences of potyviruses. Maximum homology of 56% was found in the 3' non-coding region between the petunia isolate and other potyviruses. A maximum homology of 69% was found between the amino acid sequence of the coat protein of the petunia isolate and corresponding sequences of other potyviruses. These data indicate that the petunia-infecting virus is a previously undescribed potyvirus and the name petunia flower mottle virus (PetFMV) is suggested.

An infectious viral disease of penaeid shrimp newly found in Korea.

Since 1993, massive mortalities have occurred among the penaeid shrimp Penaeus orientalis cultured in Korea. The major gross findings of the naturally occurring cases were 2 to 6 mm sized white spots on the inside of the carapace and reddish discolorization. Amphophilic to basophilic intranuclear inclusion bodies were readily observed in the epithelia of epidermis, foregut, gills, and lymphoid organs. Electron microscopy revealed enveloped and non-occluded ellipsoid to rod-shaped virus particles within the nucleus of lymphoid organ cells and the interstitial cells of the hepatopancreas of both naturally and experimentally infected shrimp. The size of the virions was 375 x 167 nm and the nucleocapsid was 290 x 75 nm. The agent which caused ectodermal and mesodermal necrosis in penaeid shrimp in Korea resembled white spot syndrome virus (WSSV) found in Taiwan with respect to gross signs, histological changes and viral morphology. Its viral morphology differed from that of the rod-shaped nuclear virus of Penaeus japonicus (RV-PJ) found in Japan. However, a 643 bp PCR product with an identical sequence to that from RV-PJ was detected. The results suggested that the Korean agent combined characters described for WSSV and RV-PJ.

Differentiation Among Garlic Viruses in Mixed Infections Based on RT-PCR Procedures and Direct Tissue Blotting Immunoassays

A total of six viruses belonging to at least three distinct genera, Potyvirus, Carlavirus, and a novel unclassified genus, were identified from infected garlic plants in Japan based on partial cDNA cloning and sequencing of genomes. We developed procedures that combined reverse-transcription polymerase chain reaction (RT-PCR) with restriction analysis for identification of each of the six viruses. The respective viral coat protein genes were expressed as fusion proteins in Escherichia coli, and the products were used as immunogens for producing antibodies that reacted against viral particles. The antisera obtained specifically recognized each of three types of rod-shaped virus particles according to immunoelectron microscopy and enzyme-linked immunosorbent assay. Furthermore, a convenient serological method based on direct tissue blotting immunoassay (DTBIA) was developed. Survey of virus incidence using both DTBIA and RT-PCR presented direct evidence for mixed infections of garlic plants with several viruses. In addition, DTBIA is suitable for large-scale and routine diagnosis of garlic viruses.

Static and Dynamic Light Scattering by Concentrated Colloidal Suspensions of Polydisperse Sterically Stabilized Boehmite Rods

Static and dynamic light-scattering experiments have been performed on isotropic disper- sions of sterically stabilized polydisperse boehmite rods, with an average length to width ratio of 6.4. Measurements were made up to concentrations close to the isotropic nematic phase separation. At high concentrations, the scattered intensity autocorrelation function of the rods is found to separate in two modes that are attributed to collective and exchange diffusion. Corresponding mode amplitudes are in fair agreement with results of the polydisperse scaled particle theory. Whereas the collective mode decay time is hardly concentration dependent, the exchange mode decay time decreases exponentially with concentration. Many experimental model studies on structural and dynamical properties of concentrated colloidal systems have been performed using static and dynamic light ~cattering.l-~ Most of these studies are devoted to spherical particles, although several experiments on rod-shaped virus particles (mostly tobacco mosaic vi- r~s)~!* and rigid rodlike polymer^^!^ have been per- formed. Here we present a static and dynamic light-scattering study on dispersions of poly(isobutene)-grafted boehmite rods, the synthesis of which was recently developed in our laboratory.lOJ1 The rods used in this study have an average length of 71 nm and an average width of 11.1 nm, with a polydispersity of about 40%. The concentration ranges from the dilute regime to the concentrated regime close to the isotropic nematic phase separation. The main feature of the dynamic light-scattering

Ultrastructural changes in salivary glands of tsetse, Glossina morsitans morsitans, infected with virus and rickettsia-like organisms

Electron microscope observations on enlarged hypertrophied salivary glands dissected from adult laboratory-reared male Glossina morsitans morsitans show a concurrent infection of the salivary gland tissue with rod-shaped virus particles and intracellular rickettsia-like organisms. The latter are found intracellular in the epithelium and in the gland lumen enclosed within lytic zones. The virus particles are found within the degenerating cytoplasm, nuclei, and lumen of the cell where they are especially numerous. Stratified epithelium and gland enlargement are a prominent feature of the infection. These observations suggest that biological associations between salivary gland tissue and diverse microbes may be more common than formerly recognized. The microbes appear to cause damage to salivary gland cells, causing hyperplasia which assumes pathologic proportions.

Purification and Partial Characterization of a Virus from Pineapple

Double-stranted RNA and long, flexuous, rod-shaped virus particles (1,200×12 nm) were recovered from mealybug wilt-affected pineapple (Ananas comosus) plants from plantations in Hawaii. A purification protocol was developed, and the virus partially characterized. Based upon its morphology the molecular weight of the coat protein, and analysis of double-stranded RNA, we tentatively assign this virus to the type II closterovirus group

Does TMV uncoat cotranslationally in vitro?

Despite extensive knowledge of the structure, assembly and disassembly of tobacco mosaic virus (TMV) and some similar, rod-shaped virus particles in vitro, we still have little information on uncoating and assembly events during the viral infection cycle in plants. A recent, surprising discovery that TMV particles will programme virus-specific protein synthesis in vitro and undergo progressive, 5′ to 3′, cotranslational disassembly, has stimulated a search for so-called ‘striposome’ complexes (80S ribosomes on the exposed RNA portion of partly uncoated virions) in infected cells.

High-voltage electron microscopy of insect capsule viruses

The High-Voltage Electron Microscope (HVEM) affords the researcher an opportunity to study insect virus inclusion bodies, intact, without resorting to physical thin-sectioning or chemical degradation procedures. In this manner polyhedral inclusion bodies, isolated from insects infected with nuclopolyhedrosis viruses (NPVs) were observed with the HVEM, and the numerous viruses lying internally, clearly delineated.In the present report we used the HVEM to study, in whole mount, another type of insect virus inclusion body, that from the capsule (granulosis) viruses of two hosts, the fall webworm, Hyphantria cunea (Drury) and the yellow wollybear, Diacrisia virginica (Fabricius). The insect capsule viruses, like the NPVs, are characterized by a crystalline protein matrix which occludes enveloped, rod-shaped virus particles. Whereas the inclusion bodies of NPVs contain a number of virus particles in each inclusion body, those of the capsule viruses contain, generally, only one virus particle in each capsule or granule.

Electronmicrofcopic Analysis of a Cabbage Moth Virus

A nucleoplyhedrosis virus (NPV) has been isolated from the cabbage moth (Mamestra brassicae, Linnaeus), a serious pest insect, and bears promise of becoming an effective biological insecticide against its host. Our studies on this virus constitute part of an effort to support its approval as an insect control agent.The NPVs are characterized by the presence of polyhedral inclusion bodies, containing rod shaped virus particles. When ingested by the host insect, the inclusion bodies are broken down in the gut. The released virus particles infect and multiply in the nuclei of susceptible cells. Viruses having these properties are representatives of the Baculoviruses, subgroup A.The inclusion bodies of the cabbage moth NPV (Darmstadt strain) analyzed were approximately 1 - 2.3 um in diameter (Fig. 1). We used the HVEM to penetrate whole mount, naturally contrasted, airdried inclusion bodies to obtain their average mass value.

Título en español.

De plantas de caña de azúcar de la variedad CP31-294 afectadas con mosaico, se extrajo jugo y se tiñó negativamente. Al examinarlo al microscopio electrónico se observaron partículas bacilares de virus. Las partículas eran de las cepas A y B y medían 720-1.446 nm x 14-15 nm y 720-1, 730 nm x 14-15 nm, respectivamente. Cincuentiuna variedades prometedoras de caña de azúcar se inocularon con una mezcla de las dos cepas en tres experimentos en invernadero durante los últimos 3 años. Las variedades PR 1141, PR 1152, PR 62-285, PR 64-1548, PR 65-153, PR 65-2538, Q 68 y la selección Soller demostraron ser potencialmente susceptibles, con una infección media de 10 a 19%. Sin embargo, ninguna de estas variedades mostraron síntomas de mosaico cuando se expusieron a la infección natural en cañaverales. Por lo tanto, como no son susceptibles al mosaico, pueden usarse en plantaciones comerciales en Puerto Rico.

Cucumber yellows virus: Its transmission by the greenhouse whitefly, Trialeurodes vaporariorum (westwood), and the yellowing disease of cucumber and muskmelon caused by the virus.

A previously undescribed disease of the yellowing type was found to occur on cucumber and muskmelon in commercial greenhouses in Kanto area. Long, flexuous, rod-shaped virus particles, 12×1, 000nm approximately, were detected in dip preparations from diseased plants of cucumber and muskmelon. The detection was not easy, because the particles were small in number and labile in nature. The particles were also observed in thin sections prepared from both naturally and artificially infected plants of cucumber and muskmelon. Their presence was always restricted to phloem cells, but sometimes they were found in xylem parenchyma cells of vascular bundles. Virus-infected cells were also characterized by the development of amorphous granular structures and small vesicles containing fibrous materials in the cytoplasm. Both of them seemed to be closely related to virus multiplicaiton, and was considered to be viroplasms. Phloem necrosis was usually observed in diseased plants. It caused a remarkable accumulation of starch grains in chloroplasts in mesophyll cells. The virus was transmitted to healthy seedlings of cucumber and muskmelon by viruliferous greenhouse whiteflies, but not by inoculation of sap. From these results, the yellowing disease of cucumber and muskmelon was concluded to be caused by the long, flexuous, rod-shaped virus, which was named cucumber yellows virus.

Electron microscope observations on Acholeplasma laidlawii viruses.

Cultures ofAcholeplasma laidlawii were inoculated with Mycoplasmatales virus-laidlawii 1 (MV-L 1) and examined by light and electron microscopy.A. laidlawii colonies that appeared collapsed or “cratered” were seen by electron microscopy to contain numerous rod-shaped virus particles, about 86 nm long and 13 nm wide, whereas these were not seen in normal-looking colonies. InfectedA. laidlawii cells were often seen, in negative stain, to be associated with tubular filaments up to 1 μm long and 13–18 nm wide which were probably derived from the limiting membranes of the cells. In thin sections, much virus was seen attached to the outside of the cells and palisade-like arrays of virus particles were found within about 25% of the cells. Helical structures up to 160 nm long and about 12 nm wide were also seen within cells of the same preparations. They may have been the nucleocapsids of another virus unrelated to MV-L 1.

Electron microscopy of soil-borne wheat mosaic virus in host cells

Electron microscopy of sections of pelleted soil-borne wheat mosaic virus and infected leaf cells of wheat ( Triticum aestivum L.) revealed hollow-cored rod-shaped virus particles in various types of aggregates. Small, loose clusters of particles were often associated with large masses of loosely clumped, convoluted tubules of about twice the diameter of virus particles. Virus particles were also peripherally associated with small crystalline inclusions occurring in host cell cytoplasm. Highly ordered aggregations of virus particles were found in vacuoles of relatively old cells. Cross sections of these aggregates showed a hexagonal arrangement of virus particles with denselv staining material between adjacent particles.

Two nuclear polyhedrosis viruses of the Douglas-fir tussock moth

Two nuclear polyhedrosis viruses affecting the Douglas-fir tussock moth, Hemerocampa pseudotsugata, are described. One induces formation of polyhedral bodies containing single rod-shaped virus particles, each within its own envelope. The other produces polyhedra with bundles of several rods within a common envelope. The same tissues of the host are affected by both viruses, although not equally. Mean lengths of the rods of the two types differ. One type of virus has been found in only a few geographic locations.

Infectious components of granulosis virus of the codling moth, Carpocapsa pomonella

Infectious elements of a granulosis virus of the codling moth, Carpocapsa pomonella, were investigated using centrifugation techniques. An infectious form, smaller than the rod-shaped virus particle, was found. This component caused typical granulosis disease when fed to first-instar, host larvae. Thus, it contained all the genetic information necessary to cause the development of rod-shaped virus particles and mature granules.

Light and electron microscopy of inclusion bodies associated with petunia ringspot virus

Petunia ringspot virus produced both amorphous and crystalline intracellular inclusions in the cells of its hosts. The crystalline inclusions were of different shapes, all belonging to the cubic system, and seemed to develop from the X bodies. They were not soluble in water nor in 1% HCl, but were soluble in weak alkali. Ultrathin sections of virus infected Vicia faba cells showed bundles of rod-shaped virus particles in the cytoplasm: the X bodies, formed of a specific material which could be related to transitional stages of virus particle formation, and also crystalline inclusions formed by virus particles. Cross sections of the crystals showed a honeycomb hexagonal structure.

Een virus inAtropa belladonna

In a bgarden at Baarn, The Netherlands,Atropa belladonna plants, grown from seed, showed symptoms similar to those described bySmith (1946, 1957) for “Belladonna mosaic”. After inoculation of solanaceous test plants with sap from diseased plants, the following species showed symptoms:Atropa belladonna L.,Capsicum annuum L.,Hyoscyamus niger L.,Nicandra physaloides Gaertn.,Nicotiana glutinosa L.,Nicotiana tabacum L. var. Samsun,Petunia hybrida andPhysalis floridana Rydb. The symptoms suggest that the virus may be identical with that described bySmith. A high virus concentration was found inHyoscyamus niger. Nicandra physaloides, Petunia hybrida, Physalis floridana, in the roots and the pericarp of diseasedAtropa plants, and also inSolanum nigrum L. andDatura stramonium L. The latter two species showed hardly any symptoms. A. low virus concentration was found inCapsicum annuum, though this plant showed severe symptoms. The dilution end point of the virus was between 10−3 and 10−4; virusinactivation occurred between 70° and 80°C. In electron micrographs the rod-shaped virus particles appeared similar to those of rattle virus. Virus could be detected in the roots of tobacco plants after the leaves had been inoculated with sap of diseasedAtropa-plants (Table 1). The reverse did not occur. Following immersion of the roots of tobacco plants in virus-containing sap these plants were potted in steamed soil. Subsequently the roots proved to be infected but the stems and leaves contained no virus. However,Atropa plants treated in the same way, did show leaf symptoms. It appeared, that the roots of young, healthy tobacco plants could become infected with virus, when grown in naturally infested soil for only tow days (Table 2). Fungus cultures isolated from diseased roots did not show any infectivity. Nematodes are probably the vectors of this virus (Sol, Van Heuvel & Seinhorst, 1960).