Single-shelled Particles Research Articles

Electrochemical Impedance Characterization of Blood Cell Suspensions-Part 2: Three-Phase Systems With Single-Shelled Particles.

The analyses presented in Part 1 are expanded to three-phase composite materials. The theory developed in Part 1 is used for analytical and numerical calculations of dielectric spectra. In this study, three-phase systems with single-shelled particles were considered. The disordered particle distribution, aligned orientation of particles, and particles placed in different lattice structures are studied. It is shown that both two-phase and three-phase composites exhibit β-dispersion, while three-phase composites additionally exhibit δ-dispersion. This is the fundamental difference in the spectra for two- and three-phase materials. In the case of aligned orientation, the effective permittivity in the direction perpendicular to the spheroid plane exhibits a maximum at a volume fraction of approximately 0.5. Both two- and three-phase materials display this behavior. This may be of interest for the development of new metamaterials. The dielectric properties of composite materials with random distribution and periodic arrangement of particles differ significantly. The dielectric spectrum of erythrocyte suspension in plasma was measured by means of electrochemical impedance spectroscopy. The measurement system consisted of a small chamber with two planar electrodes placed at the bottom and an impedance analyzer. The dielectric properties of erythrocyte cytoplasm and membrane were numerically determined based on experimental data. The measurement of the dielectric properties of whole blood and blood components is very promising for various medical applications.

Electrochemical Impedance Characterization of Blood Cell Suspensions. Part 1: Basic Theory and Application to Two-Phase Systems

Electrochemical impedance spectra of composite materials contain information on the topological arrangement, volume fraction, and shape of particles, as well as the dielectric properties of the matrix and particles. The objective of this study is to investigate how these parameters affect the dielectric spectrum and what reliable information can be extracted from experimental data. The main attention was focused on systems with dielectric behavior similar to that of human blood. Mostly plasma and erythrocytes determine the dielectric properties of whole blood. Erythrocytes suspended in plasma can be considered as three-phase systems with single-shelled particles. A theoretical approach based on the effective medium theory is developed for calculating the effective permittivity and conductivity of three-phase composites at a wide frequency range (from 0 to 1 GHz). A finite-difference method is applied to model three-dimensional periodic structures. A special case of two-phase materials is used to demonstrate the influence of the shape and arrangement of particles on dielectric properties. Theoretical and numerical approaches are applied to two-phase composites with spherical, spheroidal and biconcave particles and are compared with each other and with published data. It is shown that two-phase composites exhibit only β-dispersion. In contrast to the quasi-static limit, the wide-bandwidth impedance spectroscopy makes it possible to distinguish between disordered and regular arrangements of spheroidal and biconcave particles. The results can be used to analyze the dielectric properties of blood, which is very promising for various medical applications. This study of two-phase composites can be further extended to three-phase composites.

Simulations of graphitic nanoparticles at air-water interfaces.

The free energy associated with transferring a set of fullerene particles through a finite water layer is calculated using explicit solvent molecular dynamic simulations. Each fullerene particle is a carbon network of one or more spheroidal shells of graphitic carbon, and include single-shell (single-wall) or nested multi-shelled (nano-onions) structures ranging from 6 to 28 Å in radius. Corresponding changes in energy suggest a stronger affinity of carbon nano-onions for water compared to their single-shelled analogues. In the case of multi-shelled structures, the free energy profiles display a global minimum only in the bulk liquid indicating a high affinity of multi-shelled fullerene for complete hydration. Single-wall particles however, display a minimum at the air-water interface and for particles larger than 2 nm this minimum is a global minimum possessing a lower energy compared to the particle's state of complete hydration. While the propensity for single-shell particles to adsorb to the air-interface may increase with increasing particle size, there is an indication based on line tension calculations that larger single-shell particles may actually exhibit enhanced wetting compared to their smaller analogues.

Liposomes as a model for the study of high frequency dielectrophoresis.

Liposomes were used as a physical model to study the dielectrophoretic response of single-shelled particles at high frequencies. For a typical particle, the single-shelled theoretical model predicts a lower cross-over frequency that depends upon the dielectric properties of the shell and an upper crossover frequency that depends upon the dielectric properties of the interior. Dried liposomes were rehydrated in media with conductivity ranging from 100 to 2000 μS/cm. The high frequency dielectrophoresis response of the liposomes was observed in the range of 1-80 MHz at 30 volts peak-to-peak, and the upper cross-over frequency was recorded. The experimental results closely matched the theoretical expectations. In particular, the upper cross-over frequency ranged from 9 to 60 MHz and was found to depend linearly on the interior conductivity of the liposome. These results further confirm the single-shell model at high-frequencies. Moreover, they suggest liposomes may be a useful model particle for use during the development of dielectrophoresis-based devices.

Evolutionary genomics of mycovirus-related dsRNA viruses reveals cross-family horizontal gene transfer and evolution of diverse viral lineages

BackgroundDouble-stranded (ds) RNA fungal viruses are typically isometric single-shelled particles that are classified into three families, Totiviridae, Partitiviridae and Chrysoviridae, the members of which possess monopartite, bipartite and quadripartite genomes, respectively. Recent findings revealed that mycovirus-related dsRNA viruses are more diverse than previously recognized. Although an increasing number of viral complete genomic sequences have become available, the evolution of these diverse dsRNA viruses remains to be clarified. This is particularly so since there is little evidence for horizontal gene transfer (HGT) among dsRNA viruses.ResultsIn this study, we report the molecular properties of two novel dsRNA mycoviruses that were isolated from a field strain of Sclerotinia sclerotiorum, Sunf-M: one is a large monopartite virus representing a distinct evolutionary lineage of dsRNA viruses; the other is a new member of the family Partitiviridae. Comprehensive phylogenetic analysis and genome comparison revealed that there are at least ten monopartite, three bipartite, one tripartite and three quadripartite lineages in the known dsRNA mycoviruses and that the multipartite lineages have possibly evolved from different monopartite dsRNA viruses. Moreover, we found that homologs of the S7 Domain, characteristic of members of the genus phytoreovirus in family Reoviridae are widely distributed in diverse dsRNA viral lineages, including chrysoviruses, endornaviruses and some unclassified dsRNA mycoviruses. We further provided evidence that multiple HGT events may have occurred among these dsRNA viruses from different families.ConclusionsOur study provides an insight into the phylogeny and evolution of mycovirus-related dsRNA viruses and reveals that the occurrence of HGT between different virus species and the development of multipartite genomes during evolution are important macroevolutionary mechanisms in dsRNA viruses.

Assembly of Viroplasm and Virus-like Particles of Rotavirus by a Semliki Forest Virus Replicon

In this study we have used an expression system based on Semliki Forest virus (SFV) to study assembly and intracellular localization of certain capsid proteins of rotavirus in neurons and mammalian epithelial cells. The complete genes of vp2 (vp2A) and vp6 (vp6A) of group A rotavirus (SA-11) and gene 5 encoding vp6 (vp6C) of porcine group C rotavirus (strain Cowden/AmC-1) were inserted into an SFV expression replicon. Transfection of BHK-21 cells within vitro-made SFV transcripts resulted in a high level of expression of the heterologous genes. Cotransfection with helper RNA encoding the SFV structural proteins, but lacking the genomic RNA packing signal, resulted in production of recombinant infectious virus. Immunological and biochemical analysis revealed that vp6 was expressed to high levels in primary neurons and mammalian epithelial cells and that vp6 was retained as an authentic homotrimer, stabilized by noncovalent interactions with native antigenic determinants. Thin section electron microscopy analysis revealed that vp6 alone assembled into viroplasm-like structures in the cytoplasm. While coexpression of vp2 and vp6 of group A rotavirus resulted in formation of single-shelled-like particles, no evidence of intracellular assembly was found, suggesting that other viral proteins are required for intracellular formation of single-shelled particles. A notable observation was that the vp6 proteins of group A and C rotaviruses showed different immunofluorescence patterns in BHK-21 cells; vp6C displayed an intense punctate immunofluorescence pattern, while vp6A was characterized by a pronounced filamentous staining in close vicinity to the cytoskeleton.

Genetic Characterization of the Rotavirus Nonstructural Protein, NSP4

The rotavirus nonstructural protein NSP4 plays a role in viral assembly by acting as an intracellular receptor for single-shelled particles and assisting in the translocation of these particles across the endoplasmic reticulum. Recently, NSP4 has been implicated in rotavirus virulence and is thought to act as an enterotoxin which triggers chloride secretion by a calcium-dependent signal transduction pathway. Limited sequence analysis of NSP4 shows a well-conserved protein. To define the extent of sequence variation in the gene coding for NSP4, we have sequenced this gene from nine human rotavirus strains. These data and the analysis of additional human strains and various animal rotaviruses (bovine, simian, equine, and porcine) by Northern blot hybridization suggested that three NSP4 genotypes were present among rotavirus strains. A correlation between NSP4 genotype and VP6 subgroup was also implied. Two different NSP4 genes (which encoded distinct types of NSP4 proteins) were found among standard human rotaviruses and in strains circulating in the local community and these showed homology to cognate genes in some animal strains.

Rotavirus Stimulates IL-8 Secretion from Cultured Epithelial Cells

Rotavirus is the most important cause of severe gastroenteritis in children worldwide. We have investigated cytokine responses to rotavirus infection of cultured intestinal epithelial cells. Interleukin 8 (IL-8) is a chemotactic and cell-activating cytokine that is synthesized by epithelial cells and induced in response to bacterial enteric pathogens. Rotavirus inoculation increased IL-8 mRNA levels in cultured intestinal epithelial cells within 2 hr of infection. IL-8 secretion increased 102- to 103-fold by 8 hr postinfection. Secretion of TNFα or IL-1β, cytokines which themselves increase IL-8 secretion, was not induced by rotavirus, nor was that of TNFα, IFNα, IFNγ, or IL-6. Neutralizing antibodies to TNFα or IL-1α/β did not affect the IL-8 response. Secretion of IL-8 was dependent on an intact viral capsid, as single-shell particles were inert. Neutralizing monoclonal antibodies (vp7-specific) that do not block cell attachment did block rotavirus stimulation of IL-8 secretion, indicating that attachment to the cell surface is not a sufficient stimulus to induce IL-8. Genetically inactivated rotavirus was also effective for IL-8 induction, indicating that viral replication was not required. These data suggest that epithelial cytokine IL-8 may be an important mediator of the host response to viral gastroenteritis pathogens such as rotavirus.

A hypothesis about the mechanism of assembly of double-shelled rotavirus particles.

During double-shelled (ds) particle assembly, subviral particles [possibly single-shelled (ss) particles] acquire the outer capsid protein during their transport across the endoplasmic reticulum (ER) membrane by an exocytosis-like process, probably by a fusion-like mechanism. Fine reticular material is observed around the junction area between virus particles and the ER membrane on the cytoplasmic side of projecting ss particles, suggesting this is the site of assembly of ds particles. It is assumed that the reticular material may correspond to the hetero-oligometric complexes consisting of the non-structural glycoprotein NSP4, the structural proteins VP4 and VP7, and that both VP7 and VP4 may fold onto ss particles as a complex. On the other hand, the budding process simply serves as a vehicle to transport ss particles from the cytoplasm to the ER lumen. Thus, it is assumed that the production of protein complexes may be indispensable for virion assembly, in which NSP4 regulates VP4 folding as an ER chaperone and also the exocytosis-like or fusion-like transport systems through the ER membrane.

Identification of the nucleic acid binding domain of the rotavirus VP2 protein

The bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for double-stranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified single-shelled particles.

Topology of Bovine Rotavirus (RF Strain) VP6 Epitopes by Real-Time Biospecific Interaction Analysis

An automated biosensor system designed for measuring molecular interactions in real-time (BIAcore) was used to characterize monoclonal antibodies (Mabs) raised against the inner capsid protein (VP6) of the bovine rotavirus (RF strain). Six Mabs, all reactive in Western blot and in indirect immunofluorescence assays, were mapped, using purified recombinant VP6. These Mabs were delineated into several groups of antibodies, interactions of selected monoclonal antibodies with purified vital particles were studied by the BIAcore methodology. We showed that some Mabs did not react with single-shelled particles. Conversely, several Mabs reacted with single-shelled particles and one antibody reacted with both single-shelled and double-shelled particles. The latter Mab seemed to interact with VP6 through the holes of the outer capsid and its interaction with the double-shelled particles induced a significant decapsidation. These results allowed a better characterization of the epitopes of VP6. The localization of the epitopes in the viral particle is discussed in comparison with a pepscan study that determined the reactivity of Mabs with nested heptapeptides derived from the whole VP6 molecule.

Temperature-Sensitive Lesions in the Capsid Proteins of the Rotavirus Mutants tsF and tsG That Affect Virion Assembly

The SA11 rotavirus mutants tsF and tsG contain temperature sensitive (ts) lesions in the capsid proteins VP2 and VP6, respectively, that interfere with their ability to assemble. To understand the nature of their lesions, full-length cDNAs of tsF gene 2 and tsG gene 6 were prepared from viral mRNA by reverse transcription and polymerase chain reaction. Comparative sequence analysis indicated that the ts phenotype of tsF VP2 is due to an Ala → Asp substitution at position 387. The mutation falls outside of those regions of VP2 previously suggested to be of functional significance and therefore points to a previously unidentified site in VP2 that is important for the assembly of viral cores. Comparative sequence analysis showed that tsG VP6 contains two mutant amino acids, i.e., Thr-10 and His-13, and therefore one or both of these mutations are responsible for the ts phenotype of the mutant VP6. In the case of other group A and group C VP6 sequences, these residues are Ser and Asp, respectively. Characterization of tsG-infected cells by indirect immunofluorescence staining showed that while viroplasmic inclusions are formed at the nonpermissive temperature, the mutant VP6 accumulates in these structures only at the permissive temperature. While influencing intracellular accumulation, the Thr-10 → Ser and His-13 → Asp mutations in tsG VP6 are probably not directly involved in the interaction of VP6 with VP2, as VP6 deletion mutants lacking residues 10 and 13 retain the ability to bind VP2 in vitro . Analysis of VP6 failed to confirm previous reports that the protein was myristylated and thus excludes the possibility that this cotranslational modification is temperature-dependent for tsG VP6. Together, these data suggest that the amino terminus of VP6 plays an essential role in virus assembly in vivo, perhaps by being necessary for the movement of the protein to viroplasmic inclusions, the site of core and single-shelled particle formation.

Intracellular manipulation of disulfide bond formation in rotavirus proteins during assembly.

Rotavirus undergoes a unique mode of assembly in the rough endoplasmic reticulum (RER) of infected cells. Luminal RER proteins undergo significant cotranslational and posttranslational modifications, including disulfide bond formation. Addition of a reducing agent (dithiothreitol [DTT]) to rotavirus-infected cells did not significantly inhibit translation or disrupt established disulfide bonds in rotavirus proteins but prevented the formation of new disulfide bonds and infectious viral progeny. In DTT-treated, rotavirus-infected cells, all vp4, vp6, and ns28 epitopes but no vp7 epitopes were detected by immunohistochemical staining with a panel of monoclonal antibodies. When oxidizing conditions were reestablished in DTT-treated cells, intramolecular disulfide bonds in vp7 were rapidly and correctly established with the restoration of antigenicity, although prolonged DTT treatment led to the accumulation of permanently misfolded vp7. Electron microscopy revealed that cytosolic assembly of single-shelled particles and budding into the ER was not affected by DTT treatment but that outer capsid assembly was blocked, leading to the accumulation of single-shelled and enveloped intermediate subviral particles in the RER lumen.

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.

Inhibition of in vitro reconstitution of rotavirus transcriptionally active particles by anti-VP6 monoclonal antibodies.

Six monoclonal antibodies specific for the major capsid protein of rotavirus, VP6, previously characterized, were tested in a biological assay for their capacity to block the transcriptase activity associated with the single-shelled particles. The results showed that two MAbs (RV-50 and RV-133), specific for distinct antigenic sites, were able to block the transcription when they were incubated with a purified baculovirus-expressed group A VP6, prior to the reconstitution of the single-shelled particles from the cores, suggesting that at least two domains are involved in active single-shelled particle reconstitution. The results obtained previously from immunochemistry of synthetic peptides did not allow us to attribute this biological activity to a particular linear sequence of the protein, the domain involved being probably complex and dependent on the folding of the protein. However, the C-terminal end, which is necessary for binding into single-shelled particles could be necessary but not sufficient to restore the transcription, since neither of these two MAbs reacted significantly with peptides of this region. These two MAbs will be useful reagents to study the interactions between VP6 and the cores.

Electron microscopic evidence for budding process-independent assembly of double-shelled rotavirus particles during passage through endoplasmic reticulum membranes.

Slowing down of the maturation process of human rotavirus particles on ice allowed the clear demonstration of two different assembly pathways through the endoplasmic reticulum (ER) membrane. One was the 'enveloped' and single-shelled (ss) particle assembly pathway, in which a transient envelope is acquired through the budding of subviral particles from the cytoplasm to the ER lumen, and later these 'enveloped' particles are released as ss particles in the ER lumen. The other was a double-shelled particle assembly pathway by which subviral particles acquire the outer capsid proteins during their transport across the ER membrane.

In vitro reconstitution of rotavirus transcriptional activity using viral cores and recombinant baculovirus expressed VP6.

Purified baculovirus-expressed group A rotavirus VP6 polypeptide was shown to be active in the recovery of the transcriptase activity associated with the reconstitution of the single-shelled rotavirus particle. Recombinant VP6 polypeptide was able to restore the transcriptional activity in purified viral cores from both SA-11 and RF rotavirus strains. Recombinant group C VP 6 (Cowden strain) is capable of binding as a trimer to group A viral core particles but unable to restore the transcriptase activity, suggesting that the binding of the polypeptide to cores is not the only requirement to restore the transcriptase activity. The VP 6 group A polypeptide was shown to bind as a monomer to viral cores, indicating that trimerization of VP 6 may be not required for reconstitution of the polymerase activity.

Characterization of a Group D Rotavirus

A rotavirus isolated from the intestinal contents of commercial ring-neck pheasant chicks was determined to be a group D rotavirus based on its RNA electropherotype and its serologic relatedness to known turkey group A and D rotaviruses using immune electron microscopy and agar-gel immunodiffusion assays. Further characterization revealed a buoyant density in cesium chloride of 1.347 g/cm3 for double-shelled particles and 1.365 g/cm3 for single-shelled particles. The average size for the pheasant-origin group D rotavirus was determined to be 80 nm for double-shelled particles and 70 nm for single-shelled particles. In contrast, the average size for a turkey group A rotavirus isolate was determined to be 73 nm for double-shelled particles and 60 nm for single-shelled particles. There was no evidence of viral replication by the pheasant-origin group D rotavirus (either cytopathic effect or increase in numbers of viral particles) after six passages in MA104 cells. The hemagglutinating activity of the pheasant origin group D rotavirus was compared with that of a turkey origin group A rotavirus. Differences and similarities were noted depending upon the species of erythrocytes employed and the temperature of incubation.

A Subviral Particle Binding Domain on the Rotavirus Nonstructural Glycoprotein NS28

The single-shelled particle binding domain(s) on NS28 was examined by testing the ability of different truncated forms of NS28 to bind single-shelled particles (ssp). Deletion of amino acids (aa) 161 to 175 of NS28 abolished ssp binding activity. Deletion of the last three aa (173-175) of NS28 diminished, but did not abolish, the ligand binding activity in our assay conditions. An internal deletion of NS28 (aa 110 to 155) also significantly diminished ssp binding activity in standard binding assays. As an alternative approach to study the ssp binding domain on NS28, we mapped the epitope of binding of monoclonal antibody BA/55, which was found to block ssp binding to NS28. Immunoprecipitation experiments done with truncated mutants of NS28 located the epitope of BA/55 to aa 149-160 of NS28, immediately adjacent to or partially overlapping the putative ssp binding domain. Experiments using synthetic peptides mimicking the carboxy end of NS28, found these peptides were not able to compete for ssp binding. Together, these results suggest that the ssp binding site in NS28 (aa 161-172) is highly dependent on the conformational integrity of the cytoplasmic C-terminus of NS28. NS28 truncation mutants also were assayed for interactions with rotavirus VP4 expressed in baculovirus. Amino acids 112 to 148 of NS28 were found to be critical for NS28-VP4 binding. Unexpectedly, aa 149 to 175 not only were nonessential for interaction with VP4, but mutants lacking those aa showed improved binding activity. We hypothesize that the VP4 binding domain may be buried in the NS28 cytoplasmic domain, and that the binding of ssp and VP4 may be an interdependent process that functions in conjunction with triggering of the budding of the whole complex into the endoplasmic reticulum. These results demonstrate the pleiotropic properties of NS28 in the unique rotavirus morphogenetic process.

Purification of the IDIR Strain of Group B Rotavirus and Identification of Viral Structural Proteins

A purification scheme was developed that allowed for the partial purification of complete, double-shelled particles of the infectious diarrhea of infant rats (IDIR) virus, a group B rotavirus (GBR). Structural proteins from the isolated, complete viral particles were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and evaluated by silver staining and by immunoblotting using antisera obtained from rats that had recovered from IDIR virus infection. Analysis of the stained gels and immunoblots demonstrated the presence of IDIR virus structural proteins having estimated molecular weights of 130, 100, 88, 80, 61, 44, 32, and 25 kDa. Treatment of complete viral particles with EDTA removed the outer capsid layer producing single-shelled particles that lacked the 80, 61,32, and 25 kDa proteins seen in the double-shelled particles. The 44-kDa protein was most abundant and was recognized by a mouse monoclonal antibody and hyperimmune guinea pig serum prepared against IDIR virus and by hyperimmune guinea pig serum prepared against adult diarrhea rotavirus (ADRV), a human strain of GBR. Convalescent serum obtained from a piglet inoculated with a porcine GBR reacted with the 61 kDa, outer capsid protein of IDIR virus. This information on the structural nature of IDIR virus should be helpful in establishing the genetic and structural relationships among the GBR and to further exploit the potential of IDIR virus as a tool for understanding GBR infections in human and animal populations.