Spherical Virus-like Particles Research Articles

Virus-like Particles and Cellular Changes in Plants Infected with Sweetpotato Viruses

Studies with the transmission electron microscope were used to detect and attempt to identify viruses infecting sweetpotato (Ipomoea batatas) and other Ipomoea species. Flexuous-rods, short curved-rods, and spherical virus-like particles were observed in cells of symptomatic plants. Also, various cytopathic changes such as crystals, vesicles, fibril structures, and cylindrical inclusions were observed. The present study showed that some of these cytopathic changes were associated with some viral groups, which might be helpful in diagnosis.

Characterization of a novel satellite virus and a strain of Himetobi P virus ( Dicistroviridae) from the brown planthopper, Nilaparvata lugens

Two distinct spherical virus-like particles were purified from the brown planthopper, Nilaparvata lugens. One was a geographical isolate of Himetobi P virus ( Cripavirus, Dicistroviridae). The other was 30 nm in diameter and contained positive-stranded RNA. The RNA was 1647 nucleotides in length and encoded only its own capsid protein, indicating that this particle is a satellite virus. Transmission tests showed that the satellite was transmitted vertically; however, its helper virus was unknown. We named this satellite Nilaparvata lugens commensal X virus (NLCXV).

Japanese encephalitis subunit vaccine composed of virus-like envelope antigen particles purified from serum-free medium of a high-producer J12#26 cell clone

A stable cell clone, J12#26, which continuously secretes large amounts of the envelope (E) antigen of Japanese encephalitis (JE) virus (J. Virol. 77 (2003) 8745) was adapted to serum-free medium. The J12#26 antigen possessed hemagglutinating activity, as well as the viral E and M proteins. More than 10 and 1 mg of the antigen quantified with the licensed JE vaccine (JE-VAX) as a standard by E-ELISA and protein determination, respectively, were recovered from 500 ml of serum-free medium by membrane ultrafiltration, Sephacryl S-300 chromatography, sucrose gradient centrifugation and Sephadex G-25 chromatography. SDS-PAGE and Western blot analyses confirmed the high yield and purity of the J12#26 E antigen, which was comprised of small spherical virus-like particles (VLP) of approximately 25 nm in diameter. This antigen induced in mice without adjuvant neutralizing antibody (NT Ab) titers, as high as or higher than the licensed JE vaccine, and complete protection against challenge with wild-type virus. These results suggest that the J12#26 antigen is a promising second-generation JE subunit vaccine.

Viral encephalopathy and retinopathy of farmed marine fish species in Italy

Viral encephalopathy and retinopathy, otherwise known as fish encephalitis or viral nervous necrosis (VNN), is an emerging problem in several farmed marine fish species in various geographic areas all over the world. Since summer 1995, heavy losses affecting mainly juvenile and adult sea bass ( Dicentrarchus labrax) have been observed in several on-growing facilities in Italy. Dying fish show abnormal swimming behaviour and, at temperatures higher than 20–22°C, mortality rates range between 15 and 50%. Neither significant external nor internal gross pathological signs, except frequent abnormal swim bladder hyperinflation, were detected. Histological investigations reveal vacuolations in the grey matter of the brain and spinal cord and in the granular layers of the retina. Serial tissue sections examined by an immunohistochemical method carried out with antisera against fish nodaviruses showed a positive reaction. Additionally, spherical virus-like particles 22–25 nm in diameter were detected by electron microscopy in negative stained preparations of brain tissues, and the same samples gave a positive reverse transcription–polymerase chain reaction (RT–PCR) for the T4 region of the fish nodavirus gene. These results indicate that both juvenile and adult sea bass subject to mass mortality in Italy since summer 1995 are infected with a fish nodavirus and strongly suggest that the identified virus is the cause of the observed mortality.

Chayote Mosaic, a New Disease in Sechium edule Caused by a Tymovirus.

A sap-transmissible virus was isolated from chayote (Sechium edule) in Costa Rica. Infected plants showed chlorotic spots and rings, and blotchy mosaics, which often coalesced to give a complete mosaic and leaf deformation. By electron microscopy, spherical virus-like particles of approximately 29 nm in diameter were visible, and cytological changes associated with the chloroplasts were observed. The virus particles sedimented in sucrose density gradients as two components, a top component of empty protein shells and a bottom component of electron-dense particles. Electrophoretic analysis showed a single-stranded RNA of approximately 5.7 kb and capsid protein (CP) subunits of ∼22 kDa. The virus was identified as a member of the tymovirus group on the basis of particle morphology, size, sedimentation in sucrose gradients, cytopathological effects, and capsid protein and genome properties, and it was tentatively named chayote mosaic virus (ChMV).

Complementation of M Gene Mutants of Vesicular Stomatitis Virus by Plasmid-Derived M Protein Converts Spherical Extracellular Particles into Native Bullet Shapes

The matrix (M) protein of vesicular stomatitis virus (VSV) binds the nucleocapsid to the cytoplasmic surface of the host plasma membrane during virus assembly by budding. It also condenses the nucleocapsid into a tightly coiled nucleocapsid–M protein complex that appears to give the virion its bullet-like shape. As described here, temperature-sensitive (ts) M mutants produced two classes of membrane-containing extracellular particles at the nonpermissive temperature. These could be distinguished by sedimentation in sucrose gradients and by electron microscopy. One class contained nucleocapsids and envelope glycoprotein, but very little M protein. The other class was devoid of nucleocapsids. Most of these particles were spherical or pleiomorphic in shape as determined by electron microscopy. Expression of wild-type (wt) M protein from plasmid DNA using the vaccinia/T7 virus system did not enhance the incorporation of nucleocapsids into extracellular particles from cells coinfected with the ts M mutants but did enhance the incorporation of M protein into these particles. Electron microscopy showed that wt M protein served to impart the bullet-like shape typical of VSV virions to what would otherwise be spherical or pleiomorphic virus-like particles. These data suggest that there are two distinct processes in VSV envelope biogenesis. One process involves envelopment of the nucleocapsid and can be accomplished by the ts M mutants at the nonpermissive temperature, albeit at a low level compared to wt VSV. The other process involves conversion of virion components into the bullet-like shape and requires a function provided by wt M protein.

The Nucleotide Sequence and Genome Organization of Mushroom Bacilliform Virus: A Single-Stranded RNA Virus of Agaricus bisporus (Lange) Imbach

Mushroom bacilliform virus (MBV) is found in association with spherical virus-like particles in cultivated mushrooms (Agaricus bisporus) afflicted with La France disease. MBV possesses a monopartite ssRNA genome of positive sense and differs from the majority of characterized mycoviruses, which contain segmented dsRNA genomes. We have cloned and sequenced the MBV genome and determined that its length is 4009 nucleotides. The MBV genome contains four major and three minor open reading frames and has 5′ and 3′ noncoding regions of 60 and 250 nucleotides, respectively. The putative RNA-dependent RNA polymerase and the coat protein display homology with corresponding proteins encoded by certain plant viruses, particularly luteoviruses and carmoviruses.

Hepatitis C virus particle detected by immunoelectron microscopic study.

To clarify the morphology of hepatitis C virus (HCV), an indirect immunogold electron microscopic study was carried out on two plasma samples with high HCV RNA titres using polyclonal and monoclonal antibodies specific to the putative HCV envelope protein. Spherical virus-like particles, 55 to 65 nm in diameter with spike-like projections, were found in 1.14 to 1.16 g/ml fractions after sucrose density gradient centrifugation. These particles were found only in HCV-infected blood donors and had morphological features similar to those of flaviviruses. Moreover, these particles specifically reacted with the polyclonal and monoclonal antibodies to the putative HCV envelope protein. This is the first known report in which the morphology of the HCV particle is clearly shown.

Assembly of the Major and the Minor Capsid Protein of Human Papillomavirus Type 33 into Virus-like Particles and Tubular Structures in Insect Cells

Native virions of human papillomaviruses (HPV) can be isolated from genital lesions only in very limited amounts. Recent studies have shown that virus-like particles can be obtained by expression of the capsid proteins using vaccinia virus recombinants or the baculovirus system. We now present the first detailed characterization of virus-like particles of a human papillomavirus associated with malignant genital lesions, HPV-33, produced in high yield using the baculovirus expression system. Assembly of the major capsid protein L1 alone or together with the minor capsid protein L2 has been obtained. Both spherical virus-like particles of 50-60 nm diameter and tubular structures of either 25-30 nm or 50-60 nm diameter and variable length were extracted from nuclei of the infected insect cells. However, predominantly 50- to 60-nm spherical particles were found in the cell culture medium in long-term infections. The particles have icosahedral symmetry and a density of 1.29 g/cm 3 in cesium chloride corresponding to empty papillomavirus capsids. Immunoelectron microscopy confirmed the presence of L1 and L2 in the virus-like particles. The L2 protein seemed to be modified and was shown to be tightly associated with L1 using density gradient and sedimentation analysis. Approximately 50% of the L1 molecules are cross-linked by intermolecular disulfide bonds. This is the first example for the production of HPV-like particles containing both the major and the minor capsid protein using the baculovirus expression system.

Characterization of Two Viruses Isolated from Patchouli in Japan

Two sap-transmissible viruses were isolated from patchouli (Pogostemon patchouli) in Japan. The virus source plants showed faint mosaic, mottling, or no symptoms. In electron microscopic examinations, however, elongated (760 nm in length) or spherical (27 nm in diameter) viruslike particles were found. The spherical virus, designated patchouli mild mosaic virus (PaMMV), infected plants in seven families and was found to be serologically related to, but different from, broad bean wilt virus. The elongated virus, named patchouli mottle virus (PaMoV), had a narrower host range and was identified as a member of the Potyviridae on the basis of particle morphology, formation of cytoplasmic inclusions, and a distant serological relationship with turnip mosaic potyvirus

Uterine and placental alterations in pregnant sows associated with the porcine epidemic abortion and respiratory syndrome (PEARS).

In the winter of 1990/91 a new, economically devastating disease occurred in european pig breeding herds, characterized by late-term abortion, stillbirth and a high morbidity and mortality of suckling piglets. Because of the clinical picture the disease was named porcine epidemic and respiratory syndrome (PEARS). In this study investigations were carried out in tissues of uterus and placentae of late gestational sows (107 to 112 days of gestation) in three different groups of animals: group I = control animals (n = 2) group II = naturally infected sows (n = 12) from farms, where PEARS recently had been introduced; group III = sows (n = 2), experimentally inoculated with placental homogenates from animals of group II. Both in naturally infected as well as in experimentally infected sows a multifocal, lymphohistiocytic vasculitis and perivascular cell infiltration was observed in the endometrium and maternal part of the placenta, but not in the fetal one. In the fetomaternal unit there were multifocal microseparations of the epithelial layers present. Transmission electron microscopically spherical or oval virus-like particles of 45 to 75 mm in diameter were frequently found on the surface of endothelial cells of blood vessels in the maternal placenta, in a few cases in the intercellular channel system between uterine epithelial cells or on endothelial cells of capillaries in the fetal placenta. Serological results indicate, that an infection with Lelystad virus had occurred in naturally and experimentally infected sows and that transplacental infection was present.

Evidence for a viral aetiology of Jembrana disease in Bali cattle

Jembrana disease is an acute infectious disease of Bali cattle ( Bos javanicus) in Indonesia. High titres of the infectious agent are present in plasma during the febrile phase of the disease. The size of the agent determined by membrane filtration was between 50 and 100 nm, indicating it is a virus and not a rickettsia as previously proposed. Spherical virus-like particles of 75 to 130 nm diameter with a smooth membrane and frequently with an eccentric nucleoid were detected by electron microscopy in plasma from infected animals. The virus replicated in mononuclear cell cultures of peripheral blood origin but not in other cell cultures. The virus and the associated disease have characteristics consistent with virus in the family Retroviridae.

Viruslike particles associated with ringfleck mosaic of mountain ash and a mosaic disease of raspberry in the Bavarian Forest

Two types of spherical viruslike particles measuring ca. 25–30 nm and 80 nm in diameter, respec-tively, were found both in ringfleck mosaic affected mountain ash and adjacent raspberry bushes in the Bavarian Forest. The smaller particles were also found in symptomless mountain ash in the same forest. The sap from infected mountain ash reacted with an antiserum to tomato spotted wilt virus in agar-gel-diffusion test. No reactions were obtained with raspberry or mountain ash sap when tested against antisera to a number of small spherical plant viruses.

Biochemical and immunological characterization of a novel peptide carrier system using rotavirus VP6 particles

A system which allows for the efficient attachment of synthetic peptides to spherical virus-like particles assembled from the VP6 rotavirus nucleocapsid protein is described. This attachment was shown to be mediated by peptide-protein interactions and did not require additional chemicals for conjugation. The resulting large macromolecular structure was highly immunogenic for both the VP6 protein and the coupled peptides. The antibody response to peptides bound to VP6 particles was of higher titre and longer duration than that induced by other carriers. In addition, the response to VP6-coupled peptides was not affected by prior exposure to rotavirus and exhibited a range of immunoglobulin subclasses in the absence of an adjuvant. These data demonstrate that assembled VP6 spherical particles are useful carriers for low doses of synthetic peptides.

Expression of gag precursor protein and secretion of virus-like gag particles of HIV-2 from recombinant baculovirus-infected insect cells

A recombinant baculovirus carrying the gag gene but lacking the protease coding sequences of human immunodeficiency virus type 2 (HIV-2) has been constructed. When this recombinant baculovirus is used to infect insect cells, a high level of gag precursor protein, gag pr41, is expressed. Electron microscopy showed that the majority of gag pr41 was budding through the plasma membrane and being released into the culture medium in spherical virus-like particles with a diameter of approximately 100 nm. Metabolic labeling demonstrates that gag pr41 is myristylated. Our results demonstrated that HIV-2 gag pr41 can be assembled into virus-like particles in the absence of other HIV proteins. Rabbits immunized with purified gag pr41 particles produced high-titer antibody and Western blot analysis showed that anti- gag pr41 rabbit sera recognize p17, p24, and p55 gag proteins of HIV-1. These results show that gag pr41 particles are highly immunogenic and that gag proteins of HIV-1 and HIV-2 have similar antigenic epitopes.

Virus-like particles in erythrocytes of coho salmon ( Oncorhynchus kisutch)

Virus-like particles were found in erythrocytes of coho salmon ( Oncorhynchus kisutch) reared in seawater in Northern California, U.S.A. Erythrocytes from these salmon had numerous rod-shaped cytoplasmic inclusions, ranging in size from 1 to 2 μm. These inclusions were pink to red following Lieshman-Giemsa staining and were present in all of the erythrocytes from several fish. An examination of these erythrocytes by electron microscopy showed numerous spherical virus-like particles. The particles had a mean diameter of 106 nm (s.d. = 2.7, n = 20) and were evident within membranebound vacuoles in the cytoplasm and between the inner and outer aspects of the nuclear envelope. The particles were spherical, sometimes found budding into the cytoplasmic vacuoles, and possessed regular surface projections or spikes on a tightly fitting external envelope. The consistent size and structure of these particles and their absence in normal erythrocytes suggests they may represent a new virus not previously recognized in salmonid erythrocytes.

Discovery of a specific double-stranded RNA virus in Giardia lamblia

Nucleic acid samples purified from trophozoites of Giardia lamblia Portland I strain contain an ethidium-stainable band that comigrates with 7.0 kilobase DNA in agarose gel electrophoresis. The band was degradable by alkali, ribonuclease A and ribonuclease T 1, but the susceptibility toward the ribonucleases decreased with increasing ionic strength, suggestive of double-stranded RNA (dsRNA). This identification was confirmed by electron micrographs of the purified samples, which showed linear double-stranded structures with an estimated average length of 1.5 μm. In crude homogenates of G. lamblia, this dsRNA was protected against added ribonuclease A but disappeared upon adding sodium dodecyl sulfate or proteinase K. Differential centrifugations suggested an association of the dsRNA with the nuclear fraction, but it was freed to the 109 000 × g pelletable fraction with increasing homogenization. The dsRNA was purified by CsCl buoyant density gradient centrifugations in a distinct band with a ρ value of 1.368 g ml −1. Electron microscopy revealed spherical virus-like particles (VLP) with a diameter of 33 nm. VLP of similar shape and size were also identified in the nuclei of sectioned G. lamblia trophozoites. VLP yield a major protein with an estimated molecular weight of 66 000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. VLP are abundant in the culture media of stationary-phase G. lamblia Portland I strain and are able to infect the G. lamblia WB strain, which is free of the virus. There is no sequence homology between the dsRNA and the nuclear DNA of G. lamblia and thus no apparent integration of viral genome into host DNA.

Non-A, non-B epidemic hepatitis: visualization of virus-like particles in the stool by immune electron microscopy.

Acute-phase stool samples collected from hepatitis cases during outbreaks of water-borne epidemic hepatitis were examined by immune electron microscopy (IEM). Spherical virus-like particles (27 nm in diameter) were visualized in the stool of a hepatitis patient with serological evidence of non-A, non-B (NANB) hepatitis. The IEM demonstrated serological specificity of the antigen with the patient's own convalescent serum as well as a known pool of NANB hepatitis convalescent sera. It is suggested that these virus-like particles may be the aetiological agent of faeco -orally transmitted NANB epidemic hepatitis in India.

Small virus-like particles isolated from the leaf sheath tissues of rice plants and from the rice tarsonemid mites, Steneotarsonemus spinki smiley (Acarina, Tarsonemidae).

The spherical virus-like particles, 35nm in diameter, were isolated from the rice plants infected with rice ragged stunt, dwarf, black-streaked dwarf, grassy stunt viruses, as well as from “healthy” plants which were not inoculated with those viruses. The size and shape of the particles were also found in the dip preparations of the rice tarsonemid mites and eggs, Steneotarsonemus spinki Smiley (Acarina, Tarsonemidae). The mites were parasitic on the inside surface of the rice leaf sheaths, associating with brown discoloration. Antiserum against the 35nm particles purified from the sheaths after carefully removing the mites and eggs by brushing, reacted with the particles appeared in the dip preparations from the mites and eggs. The purified preparations of the 35nm particles showed typical absorption profile of nucleoprotein.The particles were detected by ELISA in the leaf sheaths and in the hulls of rice grains, but not in the leaves, roots and paddy water. Ultrathin sections of the leaf sheaths revealed large amount of the 35nm particles in the inner epidermis and the adjacent parenchymatous cells. This is the first report of the virus-like particles associated with the tarsonemid mites that seems to propagate in the rice plants.

Two types of virus-like particles isolated from downy mildew diseased rice plants.

Two morphologically distinct types of small, spherical virus-like particles (VLPs), tentatively designated as type A and B, were purified from rice plants infected by downy mildew in field. Type A shows a diameter of 30nm with phosphotungstic acid (PTA) staining or 32nm with uranyl acetate (UA) staining, bearing spikes of 4nm on its periphery. Type B has a diameter of 32nm with PTA staining or 35nm with UA staining and no spikes. Purified preparations of type A and B showed an ultraviolet absorption spectrum with maximum at 260nm and minimum at 244nm, indicating their nucleoproteinaceous nature. The A260/A280 ratios of type A and B were 1.44 and 1.60, respectively. Antisera prepared against type A and B individually did not react with heterologous antigens. Out of 46 samples collected from the fields throughout Miyagi Prefecture, 11 samples contained both types, 9 samples contained only type A, 21 samples contained only type B and 5 samples contained none. Healthy rice plants collected near the two diseased fields contained neither type A nor B.