Comparative ultrastructural study of four reticuloendothelias viruses.

The morphology and development of four members of the reticuloendotheliosis virus group were studied by transmission electron microscopy. Virions of duck spleen necrosis virus, duck infectious anemia virus, chicken syncytial virus, and reticuloendotheliosis virus strain T are sperical with a diameter of approximately 110 nm. They are covered with surface projections about 6 nm long and 10 nm in diameter. The center-to-center distance of surface projections is about 14 nm. The budding virions contain crescent-shaped electron-dense cores 73 nm in diameter with electron-lucent centers. After release of the virions the cores apparently become condensed to 67 nm in diameter. Virions were found budding at the plasma membrane and into smooth-walled, intracytoplasmic vesicles of productively infected cells. The distribution of budding reticuloendotheliosis viruses on cells appeared random over the cell surface, and occasionally aberrant multiple forms of budding virions were observed. The virions appear to resemble mammalian leukemia and sarcoma viruses more closely than avian leukosis-sarcoma viruses.     

Direct observation of the budding and fusion of an enveloped virus by video microscopy of viable cells

Video-enhanced microscopy and digital image processing were used to observe the assembly, budding, and fusion of Respiratory Syncytial virus. Viral filaments were seen to bud from the plasma membrane of viable infected cells to a final length of 5-10 micron with an average speed of elongation of 110-250 nm/s. The rapidity of viral assembly and its synchronous occurrence (leading to the production of several viral particles per minute from the same surface domain) suggests a directed process of recruitment of viral components to an area selected for virus maturation. Virions were also seen to adsorb to the cell surface, and to fuse with the plasma membrane. These are the first real time observations of viral morphogenesis and penetration which are crucial events in the infectious cycle of enveloped viruses.     

Studies on the budding process of a temperature-sensitive mutant of murine leukemia virus with a scanning electron microscope.

The scanning electron microscope was used to study the budding process of the wild-type Moloney murine leukemia virus and one of its temperature-sensitive mutants, designated ts 3. A considerably larger number of budding particles was observed on TB cells infected with ts 3 at the nonpermissive temperature (39 C) than at the permissive temperature (34 C). No apparent difference was noted between the number of particles on ts 3-infected cells at (34 C) and wild-type-infected cells at 34 or 39 C. Virions were detected at the cell membrane of ts 3-infected cells at 39 C as early as 8 h postinfection. Virion density increased progressively up to 48 h after which no increase was observed. An average of 1,600 virus particles was observed at the cell surface at the peak of virus production. The distribution of these on the cell membrane appeared to be random. The maximum proportion of the cell surface occupied by the viral particles did not exceed 10%. After temperature shift from 39 to 34 C, approximately 90% of the particles had dissociated from the cell membrane within 1 h.     

A new virus disease in the housefly, Musca domestica (Diptera)

Reovirus particles were isolated from adults in laboratory colonies of the housefly, Musca domestica. These particles were spherical in outline, 57–76 nm in diameter, and were found only in hemocyte cytoplasm, where virions have been disclosed by a new technique. Virions were present in large numbers, and viral inclusion bodies were identified. The virus particles had pentagonal and hexagonal shapes resembling a simple icosahedral structure. The virus was shown to be infectious and pathogenic to adult flies through injection or by feeding them suspensions from flies that had died of the virus. Electron micrographs of midgut sections from infected flies showed that the midgut cells were packed with dark undulating threads which were not present in uninfected flies. However, no virus particles or inclusion bodies could be seen in these cells. On the basis of their association with infected flies, and the similarity to results from other studies on reoviruses and insect viruses, it is suggested that these threads are an alternative replicative form of the reovirus. When the virus suspensions from heavily infected flies were dialyzed against weak alkaline solutions, the threads showed an inner component of coiled material, 12 nm in diameter, inside an envelope with a diameter of 50–83 nm, mean 60.3 ± 7.5, composed of subunits 7–8 nm long and 7–8 nm across.     

Retroviral retargeting by envelopes expressing an N-terminal binding domain.

We have engineered ecotropic Moloney murine leukemia virus-derived envelopes targeted to cell surface molecules expressed on human cells by the N-terminal insertion of polypeptides able to bind either Ram-1 phosphate transporter (the first 208 amino acids of amphotropic murine leukemia virus surface protein) or epidermal growth factor receptor (EGFR) (the 53 amino acids of EGF). Both envelopes were correctly processed and incorporated into viral particles. Virions carrying these envelopes could specifically bind the new cell surface receptors. Virions targeted to Ram-1 could infect human cells, although the efficiency was reduced compared with that of virions carrying wild-type amphotropic murine leukemia virus envelopes. The infectivity of virions targeted to EGFR was blocked at a postbinding step, and our results suggest that EGFR-bound virions were rapidly trafficked to lysosomes. These data suggest that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the correct cell compartment.     

Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles.

A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells.     

The three-dimensional structure of reovirus obtained by cryo-electron microscopy.

The structures of reovirus serotypes T2J (Jones), T3D (Dearing) and the T3D core particle have been determined by cryo-electron microscopy and image processing. At a resolution of 30 A the two serotypes have similar features. The core is visible within the virus structure. The outer surface of the virus particles contains 120 holes at T = 13.1 local 6-fold axes. The holes penetrate into the virus as far as the surface of the internal core shell. Protrusions extending 4 nm from the virus surface surround each hole on the outside of the virus. At the 5-fold axes on the surface of the virus flat 'penton craters' form covers over the underlying core spikes. The detailed structure of the reovirus shell is very different to that of rotavirus although both have holes at T = 13.1 axes. Little evidence was seen of reovirus fibres extending from the virus surface.     

A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells.

Herpes simplex virus (HSV) glycoprotein gD is a major component of the virion envelope and is thought to play an important role in the initial stages of viral infection and stimulates the production of high titers of neutralizing antibodies. We assumed that gD plays an essential role in virus replication, and so to complement viruses with mutations in the gD gene we constructed a cell line, denoted VD60, which is capable of expressing high levels of gD after infection with HSV. A recombinant virus, designated F-gD beta, in which sequences encoding gD and a nonessential glycoprotein, gI, were replaced by Escherichia coli beta-galactosidase sequences, was selected on the basis that it produced blue plaques on VD60 cell monolayers under agarose overlays containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). F-gD beta was able to replicate normally on complementing VD60 cells. However, F-gD beta was unable to form plaques on noncomplementing Vero cells. Virions lacking gD were produced in normal amounts by Vero cells infected with F-gD beta, and the virus particles were distributed throughout the cytoplasm and on the cell surface, suggesting that gD is not essential for HSV envelopment and egress. Virions lacking gD were able to bind to cells, but were unable to initiate synthesis of viral early polypeptides. Plaque production of F-gD beta particles lacking gD was enhanced by polyethylene glycol treatment, suggesting that gD is essential for penetration of HSV into cells. Other HSV glycoproteins have been implicated in the entry of virus into cells, and thus this process appears to involve multiple interactions at the cell surface.     

Replication and movement of a coat protein mutant of cymbidium ringspot tombusvirus.

The spread of cymbidium ringspot tombusvirus (CyRSV) in host tissue was studied by using a coat protein gene mutant with a six-nucleotide deletion; the deletion removes two amino acids from the shell domain (S) of the capsid protein. Mutated protein subunits were synthesized in infected cells but could not assemble into virus particles. Virions were formed, however, with inoculation of mutated RNA in transgenic plants expressing normal CyRSV coat protein. The mutant is restricted in long-distance movement in Nicotiana clevelandii, whereas it spreads systemically in N. benthamiana. These results suggest that tombusviruses may spread either as complete virions or in a nonvirion form, depending on the host plant species.     

Hard shell gas-filled contrast enhancement particles for colour Doppler ultrasound imaging of tumors

Hollow hard shell particles of 200 nm and 2 micron diameter with a 10 nm thick porous silica shell have been synthesized using polystyrene templates and a sol-gel process. The template ensures than the hollow particles are monodispersed, while the charged silica surface ensures that they remain suspended in solution for weeks. When filled with perfluorocarbon gas, the particles behave as an efficient contrast agent for colour Doppler ultrasound imaging in human breast tissue. The silica shell provides unique properties compared to conventional soft shell particles employed as ultrasound contrast agents: uniform size control, strong adsorption to tissue and cells immobilizing particles at the tissue injection site, a long imaging lifetime, and a silica surface that can be easily modified with biotargeting ligands or small molecules to adjust the surface charge and polarity.     

In vivo assembly of an archaeal virus studied with whole-cell electron cryotomography

We applied whole-cell electron cryotomography to the archaeon Sulfolobus infected by Sulfolobus turreted icosahedral virus (STIV), which belongs to the PRD1-Adeno lineage of dsDNA viruses. STIV infection induced the formation of pyramid-like protrusions with sharply defined facets on the cell surface. They had a thicker cross-section than the cytoplasmic membrane and did not contain an exterior surface protein layer (S-layer). Intrapyramidal bodies often occupied the volume of the pyramids. Mature virions, procapsids without genome cores, and partially assembled particles were identified, suggesting that the capsid and inner membrane coassemble in the cytoplasm to form a procapsid. A two-class reconstruction using a maximum likelihood algorithm demonstrated that no dramatic capsid transformation occurred upon DNA packaging. Virions tended to form tightly packed clusters or quasicrystalline arrays while procapsids mostly scattered outside or on the edges of the clusters. The study revealed vivid images of STIV assembly, maturation, and particle distribution in cell.     

Role of heterologous and homologous glycoproteins in phenotypic mixing between Sendai virus and vesicular stomatitis virus.

Phenotypic mixing between Sendai virus and vesicular stomatitis virus (VSV) or the mutant VSV ts045 was studied. Conditions were optimized for double infection, as shown by immunofluorescence microscopy. Virions from double-infected cells were separated by sequential velocity and isopycnic gradient centrifugations. Two types of particles with mixed protein compositions were found. One type was VSV particles with Sendai virus spikes, i.e., phenotypically mixed particles. A second type was Sendai virus-VSV associations, which in plaque assays also behaved as phenotypically mixed particles. The ratio of VSV G protein to Sendai virus glycoproteins on the cell surface was varied, using the VSV mutant ts045 in double infections. Thus, different amounts of the VSV G protein were allowed to reach the cell surface at 32, 38, and 39 degrees C in Sendai virus-infected cells. However, a fixed number of Sendai virus spikes was always found in the ts045 virions. This represented 12 to 16% of the number of G proteins present in normal VSV. Furthermore, the yield of ts045 virions was radically reduced during double infection when the temperature was raised to block G-protein transport to the cell surface, suggesting that the Sendai virus glycoproteins were not able to compensate for G protein in budding. These results emphasize the role of the G protein in VSV assembly.     

Characterization of adenovirus particles made by deletion mutants lacking the fiber gene.

H2dl802, H2dl807, and H5dl1021 are defective deletion mutants of human adenovirus which do not make the capsid protein fiber yet which can make substantial amounts of virus particles. Virions made by the mutants contain very little fiber (which comes from helper virus contaminants in the deletion virus stocks): less than 6% as much as that contained by wild-type virions. This demonstrates that fiber is not an essential structural component of the adenovirus virion and suggests that fiber is nonessential for virion assembly. These fiber-deficient particles are poorly adsorbed to cells, consistent with the proposed role of fiber in virus attachment. Further, virion protein precursors, including that of the virion protease, are poorly processed in these particles, suggesting a relationship between the presence of fiber and the maturation of the virus particle.     

Submicroscopic characteristics of Marburg virus and its mini genome analog replication in cell cultures

Marburg virus (Filoviridae) causes severe hemorrhagic fevers in humans and some lower primates with high mortality. The virus genome is formed by a single strand RNA of negative polarity, coding for seven structural proteins. We studied the ultrastructure of Marburg virus replicative cycle and replication of its minigenome RNA (coding for the terminal areas of the genome) in the presence of helper virus in VERO fibroblastoid cell culture and epithelioid MDCK cell culture. Ultrastructural parameters of Marburg virus multiplication in these cell cultures are virtually the same. The virus nucleocapsid assembly is performed on the outer side of EPR membrane and is not associated with preliminary accumulation of the precursor material. Virions form by budding on plasmalemma and are located on the entire surface in Vero cells and only on the basolateral surface of MDCK cells. Replication of minigenome analog of marburg virus is associated with impairment of the helper virus morphogenesis and formation of spherical pseudoviral particles.     

Speckled SiO2@Au Core–Shell Particles as Surface Enhanced Raman Scattering Probes

Silica particles of ~800 nm size were functionalized using 3-amino propyl triethoxysilane molecules on which gold particles (~20 nm size) were deposited. The resulting particles appeared to form speckled SiO₂@Au core–shell particles. The surface roughness, along with hot spots, due to nanogaps between the gold nanoparticles was responsible for the enhancement of the Raman signal of crystal violet molecules by ~3.2?×?10⁷ and by ~1.42?×?10⁸ of single-wall carbon nanotubes. It has also been observed that the electromagnetic excitation near surface plasmon resonance (SPR) of core–shell particles is more effective than off resonance SPR excitation.     

A filamentous virus of the honey bee

A common and widespread disease of honey bees, Apis mellifera, is caused by an unoccluded, Feulgen-positive, filamentous nuclear virus. Ovoid viral particles seen in diseased bee hemolymph consisted of a folded nucleocapsid within a viral envelope and were 0.40 by 0.10 μm. Virions with unfolded nucleocapsids were about 3060 by 60 nm. The disease was transmissible to bees both per os and by injection, but efforts to infect oriental cockroaches, Blatta orientalis, and the greater wax moth, Galleria mellonella, failed. The disease is apparently the same as that described as a rickettsial disease of European bees.     

Gamma Ray-Induced Small Plaque Mutants of Western Equine Encephalitis Virus

Small plaque mutants of Western equine encephalitis virus were obtained from the surviving fractions of wild-type virus which was irradiated with gamma rays. The frequency with which small plaque mutants appeared in the surviving fraction increased with the radiation dose. These mutants were not more resistant to radiation than wild-type virus. The growth rate of a mutant, S127, was lower than that of wild-type. Clonally purified mutant virions presented two peaks in a velocity sedimentation profile; peak 1 corresponded to the peak of wild type and peak 2 moved faster than peak 1. Virions of both peaks were infectious and consistently formed small plaques in chicken embryo cells. Virions reisolated from either peak and grown in chicken embryo cells also revealed two peaks in sedimentation analysis. In the electron microscope examination peak 2 proved to consist of giant form particles, each of which contained more than one nucleoid surrounded with a common envelope. Despite this remarkable morphological difference, densities of the wild-type and S127 mutant virions were similar in cesium chloride gradients. The RNAs and proteins of mutant virions could not be distinguished from those of wild type on the basis of size or charge.     

Ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment.

We reinvestigated major steps in the replicative cycle of pseudorabies virus (PrV) by electron microscopy of infected cultured cells. Virions attached to the cell surface were found in two distinct stages, with a distance of 12 to 14 nm or 6 to 8 nm between virion envelope and cell surface, respectively. After fusion of virion envelope and cell membrane, immunogold labeling using a monoclonal antibody against the envelope glycoprotein gE demonstrated a rapid drift of gE from the fusion site, indicating significant lateral movement of viral glycoproteins during or immediately after the fusion event. Naked nucleocapsids in the cytoplasm frequently appeared close to microtubules prior to transport to nuclear pores. At the nuclear pore, nucleocapsids invariably were oriented with one vertex pointing to the central granulum at a distance of about 40 nm and viral DNA appeared to be released via the vertex region into the nucleoplasm. Intranuclear maturation followed the typical herpesvirus nucleocapsid morphogenesis pathway. Regarding egress, our observations indicate that primary envelopment of nucleocapsids occurred at the inner leaflet of the nuclear membrane by budding into the perinuclear cisterna. This nuclear membrane-derived envelope exhibited a smooth surface which contrasts the envelope obtained by putative reenvelopment at tubular vesicles in the Golgi area which is characterized by distinct surface projections. Loss of the primary envelope and release of the nucleocapsid into the cytoplasm appeared to occur by fusion of envelope and outer leaflet of the nuclear membrane. Nucleocapsids were also found engulfed by both lamella of the nuclear membrane. This vesiculation process released nucleocapsids surrounded by two membranes into the cytoplasm. Our data also indicate that fusion between the two membranes then leads to release of naked nucleocapsids in the Golgi area. Egress of virions appeared to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. Our data thus support biochemical data and mutant virus studies of (i) two steps of attachment, (ii) the involvement of microtubules in the transport of nucleocapsids to the nuclear pore, and (iii) secondary envelopment in the trans-Golgi area in PrV infection.     

Properties of Johnsongrass Chlorotic Stripe Mosaic Virus

Physicochemical, biological, and cytopathological properties of Johnsongrass chlorotic stripe mosaic virus (JCSMV) found in Iran were investigated. Virus particles were polyhedral, showed a knobbed surface structure, were c. 30 nm in diameter and had a buoyant density of 1. 359 g/ml in cesium chloride. Virions contained one major protein with a molecular weight of 41 kd and a single species of ssRNA with a molecular weight of 1. 43 × 10⁶ d. Acid hydrolysis of the virus followed by thin-layer electrophoresis gave the following molar percentages of the bases: A: 23. 5, G: 27. 5, C: 26 and U: 23. Separation of nucleotides of the viral RNA using alkaline hydrolysis was not successful. Mechanical inoculation of freshly purified virus or isolated RNA failed to infect Johnsongrass or maize plants. The virus was readily detected by ELISA in seeds from infected plants and young seedlings raised from such seeds, but not in later stages of growth. Ultrathin sections of infected cells showed high concentrations of virus particles in the cytoplasm and vacuoles. Virus-like particles also occurred in the stroma of chloroplasts and mitochondria. Cisternae of endoplasmic reticulum (ER) were often extremely inflated and filled by a fine fibrillar material. Small membrane-associated vesicles were frequently found in ER elements and occurred also in the permuclear space. Based on particle structure, properties of the nucleic acid, molecular weight of the coat protein and cytopathology, the virus resembles carmoviruses. However, lack of mechanical transmissibility is not known for any virus classified with this group. No serological reaction was detected with a total of 30 antisera to carmoviruses and other isometric viruses.