Thin Sections Of Infected Cells Research Articles

Isolation and characterization of two viruses with large genome size infecting Chrysochromulina ericina (Prymnesiophyceae) and Pyramimonas orientalis (Prasinophyceae).

Two lytic viruses specific for Chrysochromulina ericina (Prymnesiophyceae) and for Pyramimonas orientalis (Prasinophyceae) were isolated from Norwegian coastal waters in June 1998. The lytic cycle was 14-19 h for both viruses; the burst size was estimated at 1800-4100 viruses per host cell for the Chrysochromulina virus and 800-1000 for the Pyramimonas virus. Thin sections of infected cells show that both viruses replicate in the cytoplasm and that they have a hexagonal cross section, indicating icosahedral symmetry. The Chrysochromulina virus had a particle size of 160 nm and a genome size of 510 kbp; the size of the major polypeptide was 73 kDa. The Pyramimonas virus had a particle size of 220 x 180 nm and a genome size of 560 kbp; the size of the major polypeptide was 44 kDa. The genome sizes of these viruses are among the largest ever reported for viruses and they are larger than the minimum required for cellular life. The Chrysochromulina virus clone CeV-01B and the Pyramimonas virus clone PoV-01B described in this study have several properties in common with other viruses infecting microalgae, suggesting that they belong to the Phycodnaviridae.

A null mutation in the gene encoding the herpes simplex virus type 1 UL37 polypeptide abrogates virus maturation.

The tegument is an integral and essential structural component of the herpes simplex virus type 1 (HSV-1) virion. The UL37 open reading frame of HSV-1 encodes a 120-kDa virion polypeptide which is a resident of the tegument. To analyze the function of the UL37-encoded polypeptide a null mutation was generated in the gene encoding this protein. In order to propagate this mutant virus, transformed cell lines that express the UL37 gene product in trans were produced. The null mutation was transferred into the virus genome using these complementing cell lines. A mutant virus designated KDeltaUL37 was isolated based on its ability to form plaques on the complementing cell line but not on nonpermissive (noncomplementing) Vero cells. This virus was unable to grow in Vero cells; therefore, UL37 encodes an essential function of the virus. The mutant virus KDeltaUL37 produced capsids containing DNA as judged by sedimentation analysis of extracts derived from infected Vero cells. Therefore, the UL37 gene product is not required for DNA cleavage or packaging. The UL37 mutant capsids were tagged with the smallest capsid protein, VP26, fused to green fluorescent protein. This fusion protein decorates the capsid shell and consequently the location of the capsid and the virus particle can be visualized in living cells. Late in infection, KDeltaUL37 capsids were observed to accumulate at the periphery of the nucleus as judged by the concentration of fluorescence around this organelle. Fluorescence was also observed in the cytoplasm in large puncta. Fluorescence at the plasma membrane, which indicated maturation and egress of virions, was observed in wild-type-infected cells but was absent in KDeltaUL37-infected cells. Ultrastructural analysis of thin sections of infected cells revealed clusters of DNA-containing capsids in the proximity of the inner nuclear membrane. Occasionally enveloped capsids were observed between the inner and outer nuclear membranes. Clusters of unenveloped capsids were also observed in the cytoplasm of KDeltaUL37-infected cells. Enveloped virions, which were observed in the cytoplasm of wild-type-infected cells, were never detected in the cytoplasm of KDeltaUL37-infected cells. Crude cell fractionation of infected cells using detergent lysis demonstrated that two-thirds of the UL37 mutant particles were associated with the nuclear fraction, unlike wild-type particles, which were predominantly in the cytoplasmic fraction. These data suggest that in the absence of UL37, the exit of capsids from the nucleus is slowed. UL37 mutant particles can participate in the initial envelopment at the nuclear membrane, although this process may be impaired in the absence of UL37. Furthermore, the naked capsids deposited in the cytoplasm are unable to progress further in the morphogenesis pathway, which suggests that UL37 is also required for egress and reenvelopment. Therefore, the UL37 gene product plays a key role in the early stages of the maturation pathway that give rise to an infectious virion.

Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney.

A long-term cell line (SHK-1) supporting replication of the causal virus of infectious salmon anaemia (ISA) has been established. The cell line was developed from a culture of Atlantic salmon (Salmo salar L.) head kidney cells. CPE was observed in SHK-1 cells 12-14 days after inoculation with ISA-infective tissue material. The time for CPE to develop decreased after repeated passages of medium from infected cell cultures to new cultures. Transmission trials demonstrated that Atlantic salmon parr developed ISA after intraperitoneal injection of preparations made from infected cells and growth medium. The ISA infectivity of the cell preparations increased with incubation time of inoculated cells. Cell cultures in a second passage were found to have a higher infectivity than the primary inoculated cultures. Virus particles with a diameter of approximately 100-120 nm, and which contained an external envelope and granules were seen in electron micrographs of thin sections of infected cells. Most of the virus particles were located extracellularly close to the cell surface, and in some cases, a connection between virus and plasma membrane could be observed. This indicates that virus particles were released by budding. Enveloped virus particles of 45-140 nm in diameter were seen in abundance in electron micrographs of a negatively stained purified virus preparation. Large, highly pleomorphic particles up to 700 nm in the longest dimension were occasionally observed in unpurified preparations. The evidence is therefore strong that the virus isolated in SHK-1 cells is the aetiological agent of ISA.

The First Record of Papaya Ringspot Virus-Type P From Australia.

Papaya ringspot virus-type P (PRSV-P) has been identified on papaya (papaw) in Australia for the first time. Outbreaks of the disease were recorded in south-east Queensland at Wamuran, Dayboro and Morayfield in the vicinity of Caboolture, in suburban Brisbane and at Beaudesert and Bundaberg during February and March 1991. Infected plants showed typical symptoms reported from other countries, including the characteristic ringspots on the fruit. Potyvirus-like particles were present in the sap of infected plants and cylindrical inclusions visible as pinwheels, bundles and scrolls were found in thin sections of infected cells. The Australian isolates of PRSV-P reacted strongly in ELlSA with antibodies to PRSV-Pand PRSV-W, but only weakly with antibodies to watermelon mosaic virus -2 or zucchini yellow mosaic virus. PRSV-P was mechanically transmitted to papaya and a number of species in the Cucurbitaceae (cucumber, pumpkin, squash, watermelon, zucchini) and was transmitted by the aphid Myzus persicae. The disease is now considered to be endemic in certain parts of south-east Queensland. A quarantine zone has been established to restrict the entry of papaya or cucurbit vegetative planting material into areas free of the disease. These measures are expected only to delay the spread of the virus.

Demonstration of cytomegalovirus and retrovirus pseudotypes in cultured guinea pig cells

A mixed viral infection with a cytomegalovirus and a retrovirus in cultured guinea pig embryo (GPE) cells was investigated. The expression of an endogenous guinea pig retrovirus (GPRV) in cultured guinea pig cells was induced by a medium containing 5-bromo-2′-deoxyuridine and dexamethasone. When the induced GPE cells were super-infected with a guinea pig cytomegalovirus (GPCMV), pseudotype virions were observed. Morphological characterization of both viruses and their locations within infected cells was achieved by examination of thin sections of infected cells with transmission electron microscopy. Immunolabeling with colloidal gold particles, 5 or 15 nm in size, permitted the identification of each virus type using GPCMV- or GPRV-specific polyclonal antibodies and the detection of a population of GPCMV and GPRV particles which expressed antigens of both viruses on their envelopes. Enhanced reverse transcriptase activity of GPRV and reduced infectivity titers of GPCMV was noted in dually infected cultures. These data suggest that interaction between GPCMV and GPRV had occurred in dually infected GPE cells and that expression of GPRV was enhanced.

A Non-capsid Protein Associated with Unencapsidated Virus RNA in Barley Infected with Barley Stripe Mosaic Virus

Summary Barley tissue with an acute systemic infection of barley stripe mosaic virus contained a large amount of unencapsidated virus RNA which was stable in extracts made in ribosome isolation buffer. The virus RNA in ribosome preparations sedimented in a broad band at 80S to 100S in sucrose gradients, which is less than the virion sedimentation rate of 180S to 200S. A protein of apparent M r 60000, which sedimented with the virus RNA, was present in ribosome extracts from infected plants but absent from those from uninfected plants. The protein is probably a virus protein because its apparent molecular weight varied slightly with the strain of virus. The structure containing the M r 60000 protein did not sediment in sucrose gradients in a compact zone as would be expected for a particle of uniform size. The M r 60000 protein was present at a concentration equal to or slightly higher (up to 400 µg/g leaf tissue) than the unencapsidated virus RNA (up to 300 µg/g leaf tissue). Sedimentation results support the conclusion that the virus RNA and the M r 60000 protein were combined in polydispersed nucleoprotein particles which may or may not have been attached to ribosomes or ribosome subunits. The M r 60000 protein was not serologically related to the capsid protein. Gold-labelled antibodies to the M r 60000 protein stained the cytoplasm in thin sections of infected cells but not that of uninfected cells. However, no distinct immunogold-labelled particle could be identified as the presumed nucleoprotein.

Intracellular localization of rotaviral proteins.

The differential distribution of two SA 11 rotaviral capsid antigens in thin sections of infected cells was examined using antibody-coated colloidal gold electron-dense particles as specific post-embedding immunocyto-chemical labels. The treatment of thin sections of conventionally fixed and embedded tissue specimens with sodium metaperiodate allowed specific localization of the antigens in tunicamycin-treated, infected CV-1 cells. Both protein antigens were investigated with specific anti-rotavirus hyper-immune sera and with specific monoclonal antibodies. These studies showed that the major outer capsid glycoprotein, gp34, of SA11 rotavirus particles was mainly located within the cisternae and along the membranes of the rough endoplasmic reticulum. The antigen of the major inner capsid protein, p42, was identified attached to enveloped virus particles, and even more obviously, on laminar crystalline structures in the nucleus and cytoplasm of the infected cells.

Amber mutants in gene 67 of phage T4: Effects on formation and shape determination of the head

Two amber mutations in gene 67 of bacteriophage T4 were constructed by oligonucleotide-directed mutagenesis and the resulting mutated genes were recombined back into the phage genome and their phenotype was studied. The 67 amK1 mutation is close to the amino terminus of the gene, and phage carrying this mutation are unable to form plaques on suppressor-negative hosts. A second mutation, 67 amK2, which lies in the middle of the gene, three codons N-terminal to a proteolytic cleavage site, produces a small number of viable phage particles. In suppressor-negative hosts, both mutants produce poly heads and proheads. 67 amK1 assembles only few proheads that have a disorganized core structure, as judged from thin sections of infected cells. The proheads and the mature phages of both mutants are mainly isometric rather than having the usual prolate shape. Depending on the 67 mutant and the host, between 20% and 73% of the particles that are produced are isometric, and 1 to 10% are two-tailed biprolate particles. 67 amK2 phages grown on a supD suppressor strain that inserts serine in place of the wild-type leucine do not contain gp67 ∗ derived from gene product 67 (gp67) by proteolytic cleavage. This demonstrates the importance of the correct amino acid at this position in the protein. Other abnormalities in these 67 amK2 phages are the presence of uncleaved scaffolding core proteins (IPIII and gp68), indicating a structural alteration in the prohead scaffold, resulting in only partial cleavage. In wild-type phages these proteins are found in the head only in the cleaved form. With double-mutants of 67 with mutations in the major shell protein gp23 no naked scaffolding cores were found, confirming the necessity of gp67 for the assembly or persistence of a “normal” core.

A Geminivirus, Serologically Related to Maize Streak Virus, from Digitaria sanguinalis from Vanuatu

Summary Electron microscopy of purified particles of a virus found in Digitaria sanguinalis from Vanuatu (formerly New Hebrides) indicated that it is a geminivirus. Preparations of virus particles contained one coat protein of mol. wt. about 27500 and circular and linear single-stranded DNA about 2350 nucleotides in length. In thin sections of infected cells, geminate particles were found in crystalline arrays both in nuclei and in the cytoplasm. The virus is serologically related to maize streak virus but differs from it with a serological differentiation index of 3.

Further observations on the morphogenesis of human rotavirus in MA 104 cells.

Human rotavirus "KUN" strain was cultivated in a fetal rhesus monkey kidney cell line, MA 104 cells. Four types of virus particles in cells infected with KUN strain were clearly identified: nucleoid cores, single-shelled particles, double-shelled particles, and membrane band, "enveloped" particles. "Enveloped" particles were found only in the thin sections of infected cells. When first visible, the virus precursors appeared at the ribosome free membrane of rough endoplasmic reticulum (RER), increasing in size while simultaneously being coated with nucleocapsid, inner shell. Single-shelled particles were also synthesized within bundles of filaments of viroplasm in the cytoplasma. During subsequent virus maturation two types of "budding" processes were observed. Double-shelled particles arising at the RER membrane entered the cisternae of the RER through an exocytosis-like process. In contrast, the "enveloped" particles developed in the cisternae by being completely enclosed with RER membrane, and later during cytolysis released the single-shelled particles. These "enveloped" virus particles appeared to be the result of inefficient virus maturation at the last stage of outer capsid formation.

Replication and morphogenesis of avian coronavirus in Vero cells and their inhibition by monensin

Avian infectious bronchitis virus (IBV) was adapted to Vero cells by serial passage. No significant inhibition of IBV replication was observed when infected Vero cells were treated with α-amanitin or actinomycin D. In thin sections of infected cells, assembly of IBV was observed at the rough endoplasmic reticulum (RER), and mature IBV particles were located in dilated cisternae of the RER as well as in smooth cytoplasmic vesicles. In addition to typical IBV particles, enveloped particles containing numerous ribosomes were identified at later times postinfection. Monensin, a sodium ionophore which blocks glycoprotein transport to plasma membranes at the level of the Golgi complex, was found to inhibit the formation of infectious IBV. In thin sections of infected Vero cells treated with the ionophore, IBV particles were located in dilated cytoplasmic vesicles, but fewer particles were found when compared to controls. A similar pattern of virus-specific proteins was detected in control or monensin-treated IBV-infected cells, which included two glycoproteins (170000 and 24000 daltons) and a polypeptide of 52000 daltons. These results suggesl lhal the ionophore inhibits assembly of a virus which malures at intracellular membranes.

Lymphocytic choriomeningitis virus. IV. Electron microscopic investigation of the virion.

The structure of lymphocytic choriomeningitis virus (LCM virus) was investigated by a variety of conventional as well as novel electron microscopic procedures. Thin sections of infected cells revealed the characteristic arenavirus entities whose interiors contain ribosome-like granules but look otherwise empty. In contrast, most thin-sectioned virus particles from infectious cell culture fluid, both untreated and highly purified with little loss of initial infectivity, appeared to be filled with rather homogeneous cores. Cores rather than granules were also found in positively contrasted whole and thin-sectioned virus particles. We favor the explanation that the sandy grains, which have given this group of viruses its name, are altered cores that happen to look like ribosomes. However, the alternative cannot yet be excluded, namely, that LCM virus-infected cells produce two types of particles, of which only the core-containing ones represent virions.

Morphogenesis of sandfly fever viruses (Bunyaviridae family)

The events occurring in the morphogenesis of sandfly fever viruses have been examined by thin-section electron microscopy and by an analysis of the association of virus-specific polypeptides with membranes of infected cells. Two representative sandfly fever viruses have been studied, Karimabad virus (KV) and Punta Toro virus (PTV), which appeared indistinguishable both in terms of virion structure, as monitored by negative staining, and morphogenesis, as observed in thin sections of infected Vero cells. Ammonium molybdate negative staining of purified, glutaraldehyde-fixed virions revealed essentially spherical particles, 87 nm in diameter, in which the surface proteins were constructed into closely packed, hollow, cylindrical subunits measuring 10–11 nm in diameter and 9–10 nm in length. These surface units are located peripherally to a 7-nm membrane bilayer which surrounds a nucleoid of variable electron density. As seen in thin sections of infected cells, the assembly of these particles was first detected at 12 hr after infection, occurred exclusively at smooth membrane vesicles, and predominantly at membranes in, or adjacent to, Golgi cisternae. Morphologically mature particles were formed by continuous involution (budding) of modified membrane segments into the lumen of these vesicles. Viral ribonucleoprotein (RNP), which was not observed free in the cytoplasm, condensed at the cytoplasmic face of these vesicles at areas at which viral spike structures could be observed at the contralateral (luminal) face. Neither RNP nor spike structures could be observed on adjacent sections of the vesicular membrane, or other membranes, which were not directly involved in the assembly of a budding virion. Analysis of the viral polypeptides in unfractionated membrane vesicles prepared from infected cells demonstrated that KV-specific envelope and nucleocapsid proteins rapidly became membrane bound, whereas a nonstructural polypeptide was found only in cytoplasmic fractions. Chymotrypsin treatment of these vesicles has indicated that at least one viral glycoprotein is inserted into cellular membranes such that approximately 12% of its sequence remains at the cytoplasmic membrane face. Proteolytic removal of this sequence generated an immunoprecipitable, glycosylated fragment which was protected from further proteolysis by its orientation within the vesicle. These morphologic and biochemical data have been used to construct a model for the assembly of these viruses. Virus particles are released from infected cells by exocytosis, a process which does not appear to result in significant modification of cell surface membranes with viral antigens.

Diagnostic Virology Using Electron Microscopic Techniques

Publisher SummaryThis chapter illustrates the development of the use of electron microscopy in viral diagnosis. The field covered is confined to medical viral diagnosis, but parallel developments have taken place in both veterinary and botanical fields and techniques derived from both these sources are also included where relevant. It is reported that the scanning transmission mode of operation, which can induce image contrast changes electronically, may enhance studies with unstained sections and perhaps facilitate thin section immune electron microscopy (IEM). The application of negative stain IEM has been particularly useful for the study of the antigenic nature of some of the newly discovered noncultivable viruses. Viral antigens can also be detected in thin sections of infected cells by IEM with suitably labeled specific antibodies. Confirmation of viral infection by electron microscopy on tissues originally processed for light microscopy is also frequently useful.

Identification of small polyhedral virus particles in thin sections of plant cells by an enzyme cytochemical technique

An enzyme cytochemical method for distinguishing particles of small polyhedral plant viruses containing single-stranded RNA from those of ribosomes in thin sections of infected cells is described. The method was completely satisfactory for plant cells infected by six of the nine viruses tested. Particles of each of these retained their encapasidated RNA and hence its densely staining properties, when aldehyde-fixed tissues were treated with RNase under conditions appropriate for the digestion of RNA from cytoplasmic ribosomes. However, in cells infected by three of the viruses studied, all of which are known to synthesize empty protein shells, enzyme treatment also removed some of the virus-encapsidated RNA. It is concluded that although the method used has considerable merit for cytopathological studies of cells infected by viruses, it should be used with caution.

Identification of rotavirus particle types.

Negative-contrast electron microscopy of purified rotavirus particles reveals two particle types: single-shelled and double-shelled particles. The relationship of these particle types, seen by negative staining, to the enveloped and various types of nonenveloped particles seen in thin sections of virus-infected cells was determined. Thin-section and negative-contrast electron microscopic analyses were performed on cell lysates from simian rotavirus. SA11-infected cells and on highly purified double- and single-shelled particles. In thin sections, double-shelled particles appeared as smooth-edged ovals containing dense nucleoids, whereas single-shelled particles had ragged edges and threads of material extending from their centers. The majority of nonenveloped particles seen in thin sections of infected cells were identified as double-shelled particles. Enveloped particles showed typical membrane structure and were observed rarely in crude rotavirus stocks, although they constitute about 10% of the particles within infected cells. It is hypothesized that the enveloped form is a transient one and the envelope is lost in the endoplasmic reticulum of the host cells. Finally, the 50-55 nm type IV particles seen within lysosome-like bodies in infected cells were identified as subviral particles formed from input virions.

Morphological phenotype of temperature-sensitive mutants of simian virus 40 in productive infection

Temperature-sensitive mutants of simian virus 40 from the complementation groups I (viral-DNA positive and V-antigen positive), II (viral-DNA positive and V-antigen negative), and III (viral-DNA negative and V-antigen negative) were examined by electron microscopy for their ability to synthesize physical virus particles at the nonpermissive temperature (40°) in productive infection. Examinations of thin sections of infected cells revealed that all the group I mutants studied were not temperature-sensitive for the synthesis of physical virus particles in terms of the proportion of cells synthesizing virions, approximate number and mode of arrangement of virions in the virus-producing cells, and morphology of virions present in the cells. Representative mutants of the complementation groups II and III were temperature-sensitive for the synthesis of virions or virion-related structures. The virus particles of the group I mutants produced at 40°, in contrast to those produced at the permissive temperature (33°), were readily destroyed after lysing the cells by repeated freezing and thawing or by sonication, but the lysate still retained V antigen, the amount of which was similar to that in lysates from the mutant-infected cells at 33° and from wild-type-infected cells at 33 and 40°. Temperature-shift experiments suggested that: (1) The event that is temperature sensitive in a group I mutant infection occurs about the time of the appearance of new infectious virions, suggesting that the mutation affects the final stage of virus maturation; (2) the affected function of the group II mutant occurs at about 10 hr prior to the time of the appearance of new infectious virions at 33°, although the mutant employed may be regarded as a late mutant because of the heat lability of the virions produced at 33° and because viral DNA synthesis is normal at 40°; and (3) the group III mutant temperature presented suggest that the three genetically distinct groups of mutants represent three functionally distinct processes in the replicative cycle of simian virus 40.

Recognition and measurement of small isometric virus particles in thin sections

By using turnip yellow mosaic virus (TYMV) as a model, the problem of determining the diameter of small isometric viruses from the interparticle distances measured in crystalline arrays is examined. The patterns to be expected for nucleoprotein particles and for empty protein shells are described and illustrated. The problem of distinguishing between these two types of particle in thin sections of infected cells is discussed. In thin sections of crystals of purified TYMV, the measured interparticle distance is always less than the diameter of the particle. In thin sections of crystalline arrays within infected cells, the interparticle distance is usually larger than expected. This is presumably because cellular components become trapped within the arrays.

The core particles of herpes simplex virus.

Numerous core particles measuring 45–50 nm in diameter were observed in the nuclei of cells infected with herpes simplex virus for 20 h. The sites for core-particle assembly appeared to be closely associated with viral DNA-replicating areas in the nucleus. The core particles were subsequently observed to be associated with the protein subunits that comprise the outer capsid of the virus. Outer capsids were formed only in areas in which core particles were present; however, numerous empty capsids (lacking a core particle) measuring 80–90 nm in diameter were also observed. The effect of trypsin digestion on thin sections of infected cells revealed that the core particles were selectively degraded by trypsin, but that the outer capsid and envelope remained relatively intact. The core particles appeared to be more sensitive to trypsin treatment after acquisition of a capsid and envelope.

Characterization of a nocardiophage for Nocardia restrictus.

Summary A nocardiophage, called Ri, was isolated from soil samples. It lysed Nocardia restrictus and a few other species of Nocardia, but none of the eight streptomycetes tested. On N. restrictus phage Ri produced plaques very irregular in shape, size and turbidity; replacement of NaCl by Ca(NO3)2 in the medium increased the uniformity of plaque morphology and gave higher counts. Electron microscope examination of the phage particles revealed a hexagonal head, 75 nm. in diameter, and a long, flexible, non-contractile tail, 330 × 10 nm., bearing a round terminal plate; a collar was sometimes visible in particles with empty heads. The phage nucleic acid is most probably double-stranded DNA. Maximum viability of phage was at pH 7; the survival was less than 0·001 % for an exposure of 30 min. at pH 5; about 20% survived at pH 6, and 10% at pH 8. When exposed to 55° or to ultraviolet irradiation, phage Ri showed similar survival curves, and was very sensitive to both treatments. Adsorption of phage to its host was rapid and efficient (80% in 5 min.); the latent period was 28 min., the rise period occurred between 28 and 37 min., and the burst size was 61. Mutation of N. restrictus to resistance to phage Ri was very rare; the apparent mutation rate was 3·4 × 10−11, which can be partly attributed to the lower growth rate of the resistant mutants. In chromium-shadowed preparations, N. restrictus appeared as short rods, 1·3 × 0·5 μ., dividing by transverse fission, showing no lateral branching characteristic of the genus Nocardia, and very few chains (never longer than 2 or 3 cells); thin sections of infected cells showed a few intracellular phage particles.