Morphological and Cytochemical Studies on Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis (LCM) virus was observed by electron microscopy in thin sections of infected tissue cultures. The particles were pleomorphic and varied greatly in size. The smaller particles (50 to 200 nm) appeared to be spherical, whereas the largest (over 200 nm) were often cup-shaped. All particles contained one to eight or more electron-dense granules which were removed by ribonuclease. The particles were formed by budding from the plasma membrane and appeared to have spikes. The morphological evidence suggests that LCM should be considered as belonging to the presently unclassified group of lipoprotein-enveloped ribonucleic acid viruses.     

Contamination of exosome preparations,isolated from biological fluids

The aim of this study was to attract attention of researchers to the problem of contamination of exosome preparations. Using a transmission electron microscope JEM-1400 (JEOL, Japan) we have examined exosome preparations, isolated according to the conventional scheme of sequential centrifugation from different biological fluids: blood plasma and urine of healthy persons and patients with oncologic diseases, bovine serum, and conditioned cell culture medium (MDCK, MDA-MB, and MCF-7 cells). All examined preparations (over 200) contained exosomes, which were identified by immuno-electron microscopy using antibodies to tetraspanins CD63 or CD9. Besides exosomes, all the studied preparations were characterized by the presence of contaminating structures: low electron density particles without limiting membrane and therefore could not be attributed to exosomes (“non-vesicles”). Two main types of the “non-vesicles” were found in the exosome preparations: particles of 20–40 nm in size, representing 10–40% of all structures in the exosome preparations; and particles of 40–100 nm in size (identical to exosomes by size). Morphology of the “non-vesicles” corresponded to that of intermediate and low density lipoproteins (20–40 nm), and very low density lipoproteins (40–100 nm), which were identical to exosomes in their size. The highest level of the contamination was detected in exosome preparations, isolated from blood samples. The results of our study indicate the need to control the composition of exosome preparations by electron microscopy and to take into consideration the presence of contaminating structures in the analysis of experimental data.     

Native hepatitis B virions and capsids visualized by electron cryomicroscopy

Hepatitis B virus (HBV) infects more than 350 million people, of which one million will die every year. The infectious virion is an enveloped capsid containing the viral polymerase and double-stranded DNA genome. The structure of the capsid assembled in vitro from expressed core protein has been studied intensively. However, little is known about the structure and assembly of native capsids present in infected cells, and even less is known about the structure of mature virions. We used electron cryomicroscopy (cryo-EM) and image analysis to examine HBV virions (Dane particles) isolated from patient serum and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice. Both types of capsids assembled as icosahedral particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope were attributed to surface glycoproteins. Their packing was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.     

Association of different kinds of bacilliform particle with vein chlorosis and mosaic diseases of raspberry (Rubus idaeus)

Thin sections of diseased raspberry (Rubus idaeus) were examined by electron microscopy. Plants of the cv. Baumforth's B and of an aphid (Amphorophora rubi)-resistant breeding selection (6820/54), both infected with raspberry vein chlorosis virus (RVCV) but not with other detectable viruses, contained large bacilliform particles c. 430 × 65 nm. Particles occurred in the cytoplasm and perinuclear space of a small proportion of xylem parenchyma cells. They had an inner core c. 25–30 nm in diameter with cross-banding of periodicity 4·5 nm, and were bounded by an outer membrane. They are probably the particles of RVCV. Plants of cv. Mailing Jewel and of a selection (M14) both showing symptoms of raspberry mosaic (veinbanding) disease contained smaller bacilliform particles c. 125 × 30 nm, which occurred singly or in clusters in the cytoplasm of a small proportion of vascular parenchyma cells. It is not known which, if any, of the viruses associated with raspberry mosaic are represented by the particles.     

Morphological Variants of Coliphage P1

Lysates of P1 from all hosts tested contained at least three morphological variants with respect to head size. These were termed "big" (P1B), "small" (P1S), and "minute" (P1M). Since successive clonings of plaques isolated on many different hosts failed to change the proportions of the variants, we concluded that the production of variants was a function of the P1 genome rather than that of the host. In the electron microscope, the heads appeared to be icosadeltahedra, having face-to-face head diameters of 86 +/- 2 nm, 65 +/- 2 nm, and 47 +/- 2 nm. Assuming the head capsids to be composed of the same protein subunits, these diameters were compatible with T = 16, 9, and 4 with a lattice constant (intercapsomere distance) of 12 to 13 nm. The tails of all variants were morphologically indistinguishable. Each consisted of a hollow tail tube surrounded by a contractile sheath attached to the head by means of a "head-neck connector" which could be a specialized vertex capsomere. In CsCl gradients, a number of bands were observed. One band contained the majority of P1B particles and 99% of the plaque-forming units. Two other bands contained P1S particles whose densities suggested a content of about 40 and 60% of the complete P1B genome. The less dense of these two bands also contained defective P1B particles with a calculated content of only 60% of the complete genome. The P1S particles tested injected their deoxyribonucleic acid (DNA) into host cells and killed them. Genetic markers contained in this band could be rescued by infectious P1B particles, confirming the evidence of Ikeda and Tomizawa that this fraction contains P1 DNA.     

Screening of Natural Waters for Viruses Which Infect Chlorella Cells

By using a plaque assay with the unicellular green alga Chlorella sp. strain NC64A as a host, viruses were screened from natural pond waters collected in Kyoto and Higashi-Hiroshima, Japan. From some samples tested, two kinds of plaques, large ( = 6 to 10 mm) and small ( = 2 to 3 mm), were detected with various frequencies. The frequency of plaques in each of the water sources was seasonal; generally, it reached a peak value (8,000 PFU/ml) in May and gradually decreased to the limit of detection (<1) in November before increasing again in early spring. Electron microscopy revealed that the purified and negatively stained viruses were very large (125 to 200 nm) icosahedral particles. The genome isolated from these particles was always a linear double-stranded DNA of 340 to 370 kbp. Electrophoresis patterns of the DNA fragments produced by digestion with restriction enzymes differed considerably from plaque to plaque, even for plaques from the same water source. However, Southern hybridization showed strong homology among all of the virus DNAs tested, indicating relatedness of those viruses. A possible use of the Chlorella virus assay system to monitor the natural population of algal cells and water quality is discussed.     

Encapsulation and crystallization of Prussian blue nanoparticles by cowpea chlorotic mottle virus capsids

Cowpea chlorotic mottle virus (CCMV) capsids were used to encapsulate Prussian blue (PB) particles based on electrostatic interaction. A negatively-charged metal complex, hexacyanoferrate (III), was entrapped inside the capsids through the disassembly/reassembly process under a pH change from 7.5 to 5.2. The loaded capsids reacted with a second Fe(II) to fabricate PB particles. The synthesis of PB in CCMV capsids was confirmed by a unique colour transition at 710 nm and by size-exclusion FPLC. Transmission electron microscopy images of PB-CCMV biohybrids presented discrete spherical particles with a relatively homogeneous size. Dynamic light scattering of PB-CCMV showed two peaks of 29.2 ± 1.7 nm corresponding to triangulation number T = 3 particles, and 17.5 ± 1.2 nm of pseudo T = 2 particles. The encapsulation and crystallization of PB in CCMV provided an efficient method for the self-organization of bimetallic nanoparticles.     

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.     

Antiviral effects of black raspberry (Rubus coreanus) seed extract and its polyphenolic compounds on norovirus surrogates

Black raspberry seeds, a byproduct of wine and juice production, contain large quantities of polyphenolic compounds. The antiviral effects of black raspberry seed extract (RCS) and its fraction with molecular weight less than 1 kDa (RCS-F1) were examined against food-borne viral surrogates, murine norovirus-1 (MNV-1) and feline calicivirus-F9 (FCV-F9). The maximal antiviral effect was achieved when RCS or RCS-F1 was added simultaneously to cells with MNV-1 or FCV-F9, reaching complete inhibition at 0.1–1 mg/mL. Transmission electron microscopy (TEM) images showed enlarged viral capsids or disruption (from 35 nm to up to 100 nm) by RCS-F1. Our results thus suggest that RCS-F1 can interfere with the attachment of viral surface protein to host cells. Further, two polyphenolic compounds derived from RCS-F1, cyanidin-3-glucoside (C3G) and gallic acid, identified by liquid chromatography-tandem mass spectrometry, showed inhibitory effects against the viruses. C3G was suggested to bind to MNV-1 RNA polymerase and to enlarge viral capsids using differential scanning fluorimetry and TEM, respectively.     

Three-dimensional structure of vaccinia virus-produced human papillomavirus type 1 capsids.

The capsid proteins of papillomavirus self-assemble to form empty capsids or virus-like particles that appear quite similar to naturally occurring virions by conventional electron microscopy. To characterize such virus-like particles more fully, cryoelectron microscopy and image analysis techniques were used to generate three-dimensional reconstructions of capsids produced by vaccinia virus recombinants (V capsids) that expressed human papillomavirus type 1 L1 protein only or both L1 and L2 proteins. All V capsids had 72 pentameric capsomers arranged on a T = 7 icosahedral lattice. Each particle (approximately 60 nm in diameter) consisted of an approximately 2-nm-thick shell of protein with a radius of 22 nm with capsomers that extend approximately 6 nm from the shell. At a resolution of 3.5 nm, both V capsid structures appear identical to the capsid structure of native human papillomavirus type 1 (T. S. Baker, W. W. Newcomb, N. H. Olson, L. M. Cowsert, C. Olson, and J. C. Brown, Biophys. J. 60:1445-1456, 1991), thus implying that expressed and native capsids are structurally equivalent.     

Intracellular hepadnavirus nucleocapsids are selected for secretion by envelope protein-independent membrane binding

Hepadnaviruses are DNA viruses but, as pararetroviruses, their morphogenesis initiates with the encapsidation of an RNA pregenome, and these viruses have therefore evolved mechanisms to exclude nucleocapsids that contain incompletely matured genomes from participating in budding and secretion. We provide here evidence that binding of hepadnavirus core particles from the cytosol to their target membranes is a distinct step in morphogenesis, discriminating among different populations of intracellular capsids. Using the duck hepatitis B virus (DHBV) and a flotation assay, we found about half of the intracellular capsids to be membrane associated due to an intrinsic membrane-binding affinity. In contrast to free cytosolic capsids, this subpopulation contained largely mature, double-stranded DNA genomes and lacked core protein hyperphosphorylation, both features characteristic for secreted virions. Against expectation, however, the selective membrane attachment observed did not require the presence of the large DHBV envelope protein, which has been considered to be crucial for nucleocapsid-membrane interaction. Furthermore, removal of surface-exposed phosphate residues from nonfloating capsids by itself did not suffice to confer membrane affinity and, finally, hyperphosphorylation was absent from nonenveloped nucleocapsids that were released from DHBV-transfected cells. Collectively, these observations argue for a model in which nucleocapsid maturation, involving the viral genome, capsid structure, and capsid dephosphorylation, leads to the exposure of a membrane-binding signal as a step crucial for selecting the matured nucleocapsid to be incorporated into the capsid-independent budding of virus particles.     

Changes in the size of reconstituted high density lipoproteins during incubation with cholesteryl ester transfer protein: the role of apolipoproteins.

It has been reported previously that the particle size distribution of discoidal, reconstituted HDL (r-HDL) changes dramatically during incubation in vitro with cholesteryl ester transfer protein (CETP). The present study was undertaken in order to determine whether these changes are influenced by the apolipoprotein composition of the r-HDL. Two preparations of r-HDL that contained egg phosphatidylcholine (egg PC) and unesterified cholesterol (UC) but differed in their apolipoprotein composition were used for the study. One preparation contained apolipoprotein (apo) A-I only (A-I w/o A-II r-HDL) while the other contained apoA-I and apoA-II (A-I w A-II r-HDL). The Stokes' radius of the major population of particles in the (A-I w/o A-II) and (A-I w A-II) r-HDL was, respectively, 4.8 and 4.9 nm. When the (A-I w/o A-II) r-HDL were incubated with CETP, most of the particles of radius 4.8 nm were converted to populations of smaller and larger particles. The smaller particles had Stokes' radii of 4.3 and 3.9 nm. The radii of the larger particles ranged from 8.2 to 13.7 nm. When the (A-I w A-II) r-HDL were incubated with CETP larger particles (Stokes' radii = 8.4-11.0 nm) appeared but there was minimal conversion to smaller particles. In addition, a significant proportion of the original (A-I w A-II) r-HDL of radius 4.9 nm was still present at the end of the incubation. These results are consistent with apoA-II inhibiting the conversion of r-HDL to small particles. It is concluded that the apolipoprotein content of r-HDL is an important determinant of the sizes of the particles that are formed during incubation with CETP.     

Novel monoclonal antibodies broadly reactive to human recombinant sapovirus‐like particles

Sapovirus (SaV), a member of the family Caliciviridae, is an important cause of acute epidemic gastroenteritis in humans. Human SaV is genetically and antigenically diverse and can be classified into four genogroups (GI, GII, GIV, and GV) and 16 genotypes (7 GI [GI.1–7], 7 GII, [GII.1–7], 1 GIV and 1 GV), based on capsid sequence similarities. Monoclonal antibodies (MAbs) are powerful tools for examining viruses and proteins. PAI myeloma cells were fused with spleen cells from mice immunized with a single type of recombinant human SaV virus‐like particles (VLPs) (GI.1, GI.5, GI.6, GII.3, GIV, or GV). Sixty‐five hybrid clones producing MAbs were obtained. Twenty‐four MAbs were characterized by ELISA, according to their cross‐reactivity to each VLP (GI.1, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GIV, and GV). The MAbs were classified by this method into: (i) MAbs broadly cross‐reactive to all GI, GII, GIV and GV strains; (ii) those reactive in a genogroup‐specific; and (iii) those reactive in a genotype‐specific manner. Further analysis of three broadly cross‐reactive MAbs with a competitive ELISA demonstrated that at least two different common epitopes are located on the capsid protein of human SaVs in the four genogroups. The MAbs generated and characterized in this study will be useful tools for further study of the antigenic and structural topography of the human SaV virion and for developing new diagnostic assays for human SaV.     

Diffusion of macromolecules and virus-like particles in human cervical mucus

To determine whether or not large macromolecules and viruses can diffuse through mucus, we observed the motion of proteins, microspheres, and viruses in fresh samples of human cervical mucus using fluorescent recovery after photobleaching and multiple image photography. Two capsid virus-like particles, human papilloma virus (55 nm, approximately 20,000 kDa) and Norwalk virus (38 nm, approximately 10,000 kDa), as well as most of the globular proteins tested (15-650 kDa) diffused as rapidly in mucus as in saline. Electron microscopy of cervical mucus confirmed that the mesh spacing between mucin fibers is large enough (20-200 nm) for small viruses to diffuse essentially unhindered through mucus. In contrast, herpes simplex virus (180 nm) colocalized with strands of thick mucus, suggesting that herpes simplex virus, unlike the capsid virus particles, makes low-affinity bonds with mucins. Polystyrene microspheres (59-1000 nm) bound more tightly to mucins, bundling them into thick cables. Although immunoglobulins are too small to be slowed by the mesh spacing between mucins, diffusion by IgM was slowed by mucus. Diffusion by IgM-Fc(5 mu), the Fc pentamer core of an IgM with all 10 Fab moieties removed, was comparably slowed by mucus. This suggests that the Fc moieties of antibodies make low-affinity bonds with mucins.     

Incomplete and Aberrant Particles of Actinophage MSP2 from Lysates of Streptomyces venezuelae

Lysates of actinophage MSP2, propagated on Streptomyces venezuelae S13, contain at least 10(11) PFU/ml. During purification by centrifugation methods and by adsorption chromatography, a number of types of aberrant and incomplete phage particles were seen by electron microscopy. Infectious MSP2 had a buoyant density in CsCl of 1.52 g/cm(3) and an absorbance at 260 nm relative to that at 280 nm (A(260)/A(280)) of 1.53. Empty capsids banded at 1.276 g/cm(3) and partially filled capsids banded at 1.351 g/cm(3), and A(260)/A(280) ratios were 0.77 and 1.24, respectively. Two kinds of light capsids found in CsCl fractions of 1.278 g/cm(3) probably include the 1.276 component. Some capsids were joined by tail-like structures. Ghosts and polyheads also were present. Aberrant particles observed by electron microscopy included two-tailed actinophage, phage with abnormal tail positions, and large-headed phage.     

Black Sea algal viruses

Monitoring of the Black Sea algal viruses in Sevastopol bays and Crimean water areas has been carried out since 2002. Based on the methods that were developed and patented by the author, more than 200 strains of algal viruses of five species of microalgae that are new to science were isolated: TvV (Tetraselmis viridis virus), DvV (Dunaliella viridis virus), PtV (Phaeodactylum tricornutum virus), PpV (Prorocentrum pusillum virus) and IgV (Isochrysis galbana virus). For the first time in the Black Sea, the Emiliania huxleyi virus (EhV) of microalgae was isolated. Using the method of electron microscopy, the Black Sea algal viruses were identified as icosahedral virions with respective sizes of 56–60, 45–48, 50–53, 88–92, and 128–132 nm, for the TvV, PtV, DvV, PpV and IgV viruses. The EhV size, as determined by the method of filtration, was within the range of 50–200 nm. In the IgV and EhV viruses we revealed a viral envelope. Based on their characters the isolated algal viruses were attributed to the Phycodnaeviridae. The maximum number of algal viruses was observed in the spring and autumn seasons, which is typical for their host phytoplankton species. The Black Sea algal viruses, TvV, PpV, IgV, and EhV, displayed no strict species specificity and have a wide range of available hosts.     

Soil chromium bioremediation: Synergic activity of actinobacteria and plants

The aim of this work was to evaluate a strategy to reduce the bioavailable chromium fraction in soil, using a Cr(VI) resistant microorganism, Streptomyces sp. MC1, under non sterile conditions, with maize plants as bioindicator and/or bioremediator.Soil samples were contaminated with 100, 200 and 400 mg kg⁻¹ of Cr(VI) or Cr(III). Bioavailable chromium (35%) was only detected in samples with Cr(VI). Soil samples with Cr(VI) 200 mg kg⁻¹ were inoculated with Streptomyces sp. MC1, and bioavailable chromium decreased up to 73%.Zea mays seedlings were planted in soil samples contaminated with chromium. Plantlets accumulated chromium mainly as Cr(III), and biomass decreased up to 88%. Streptomyces sp. MC1 was inoculated in soil samples contaminated with 200 mg kg⁻¹ of Cr(VI) and Z.mays seedlings were planted.Streptomyces sp. MC1 caused Z.mays biomass increase (57%), chromium accumulation and bioavailable chromium decreased up to 46% and 96%, respectively.This work constitutes the first contribution of cooperative action between actinobacteria and Z.mays in the bioremediation of Cr(VI) contaminated soil. The large removal capacity of bioavailable chromium by Streptomyces sp. MC1 and Z.mays infers that they could be successfully applied together in bioremediation of soils contaminated with Cr(VI).     

Membrane-associated particle aggregates in extracts of plant: infected with some viruses of the potato Y group

Membrane-associated aggregates of filamentous virus particles were found occasionally in sap from plants infected with each of five distinct viruses of the potato Y group. The aggregates measured up to 960 × 700 nm and contained from four to twenty-four virus particles. Their occurrence is apparently specifically associated with potato Y and allied viruses, observations suggesting that they may be either fragments of virus-containing cytoplasmic strands or groups of particles enveloped during cellular disruption by pieces of tonoplast with which they are associated in vivo.     

Comparison and characterization of maize stripe and maize line viruses

Two morphologically similar viruses isolated from maize in East Africa induced two distinct symptom types in maize. One, designated maize stripe virus (MSV), showed broad yellow stripes or a yellowing of the entire leaf, acute bending of the shoot apex and severe stunting. The second, maize line virus (MLV), induced continuous, narrow yellow lines along the leaf veins and did not cause apical bending or stunting. MSV and MLV were both transmitted by Peregrinus maidis (Delphacidae), but not by Cicadulina mbila (Jassidae) or by sap inoculation. Both viruses were purified by extracting systemically infected leaves in 0–5 M sodium citrate buffer and clarifying with 7 ml n-butanol/100 ml extract, followed by differential, and finally sucrose density gradient, centrifugation. Partially purified preparations of both viruses contained isometric viruslike particles of two sizes: MSV particles were 35 and 40 nm in diameter with sedimentation coefficients (so₂o, w) ^I09 ^anI0o respectively; MLV particles were 28 and 34 nm in diameter. Antisera prepared against MSV and MLV had dilution end points of 1/128 and 1/64 respectively in agar-gel diffusion tests. In tests with low-titre antisera, MSV did not react with MLV antiserum and MLV did not react with MSV antisreum; in the presence of antiserum containing antibodies to both MSV and MLV, the two viruses formed precipitin bands which crossed in the pattern of non-identity. Maize streak virus and maize mottle virus showed no serological relationship with MSV or MLV. On the basis of particle size and serology MSV and MLV are shown to be two distinct and possibly unrelated viruses. MSV and MLV apparently are dissimilar from any characterized viruses of the Gramineae.     

Herpes Simplex Virus and Human Cytomegalovirus Replication in WI-38 Cells II. An Ultrastructural Study of Viral Penetration

An electron microscope study was carried out on the early minutes of herpes simplex virus (HSV) and cytomegalovirus (CMV) penetration into WI-38 cells. Both HSV and CMV entered cells either by fusion of the viral envelope with a limiting cell membrane, or via phagocytosis. Both fusion and phagocytosis occurred within 3 min after the initiation of penetration. After fusion, the naked capsids of CMV free in the cytoplasm became coated with a fine, fibrillar material. CMV capsids thus coated retained a well-defined and easily identifiable morphology until the eclipse of visible viral particles between 1 and 1.5 days postinfection. In contrast, naked HSV capsids free in the cytoplasm were never coated. Rather, within minutes after penetration, they assumed a rounded, less regular outline, and were no longer detectable by 90 to 120 min postinfection. The free naked capsids of both viruses appeared to migrate across the cytoplasm toward the nucleus and to become located near nuclear pores. Both HSV and CMV capsids reached the nucleus as early as 5 min after the initiation of penetration. No further interaction with the nucleus could be documented. Particles were also consistently identified in the Golgi region. Phagocytosed particles generally remained within phagosomes, where they appeared to be degraded. However, stages were identified in what is believed to be the escape of enveloped viruses from phagosomes into the cytoplasm via fusion of their envelope with the phagosomal membrane.