Viral-specific DNA synthesis of Tipula iridescent virus (TIV) was not affected in Estigmene acrea cells which were continuously exposed to 300 μg/mL hydroxyurea (HU) as detected by light microscope autoradiography. Electron microscopy of such cells showed viroplasmic centres with virions in various stages of development. In nor mal cells similarly exposed to HU, nuclear DNA synthesis was reduced by 70–80%.

Comparative studies were carried out using two different insect cell lines, Aedes albopictus and Estigmene acrea, for Tipula iridescent virus (TIV) propagation. Light microscope autoradiography showed viral DNA present in viroplasmic centers (VCs) and an inhibition of nuclear DNA synthesis. These VCs appeared to be morphologically similar in both cell lines when examined by light and electron microscopy. Radiolabeled cDNA was synthesized from RNA samples obtained from infected cells at different times after infection and hybridized to TIV DNA digested with various restriction endonucleases. The results indicated that the pattern of transcription and the kinetics of TIV infection were qualitatively similar in both cell lines. The major TIV DNA components, L (greater than 174 kbp) and S1 (10.8 kbp) that are found in virions in approximately equivalent amounts, were made in both infected cell lines. However, the infected cell lines produced S1 DNA at higher levels relative to L than in virions. The cDNA hybridization studies also revealed that the S1 DNA has sequences that are transcribed and are TIV specific. While VC morphology, levels of L and S1 DNA synthesis, transcription, and capsid protein synthesis were similar in both cell lines, time course electron microscope studies revealed that progeny virions were detected only in the VCs of E. acrea cells and not in the VCs of A. albopictus cells, even by 96 hr p.i. These data suggest that the A. albopictus C6/36 cell line is semipermissive for TIV replication.

We have examined the role of cytoskeletal elements with respect to the formation and maintenance of viroplasmic centers (VCs) in Tipula iridescent virus (TIV)-infected mosquito Aedes albopictus (C6/36) cells. Filamentous systems consisting of microtubules and microfilaments were detected by immunofluorescence microscopy. Inoculation of cells with TIV resulted in an alteration of microtubule and microfilament organization whether or not VCs developed. The formation of short arrays of microtubules induced by taxol or the depolymerization of microtubules by colchicine, as observed by immunofluorescence microscopy, had no apparent effect upon the development of VCs as detected by Hoechst staining and electron microscopy. The dissolution of the actin-containing filamentous system by cytochalasin B also had no effect upon development. We conclude from these results that microtubules and microfilaments are not involved in the formation or maintenance of VCs in TIV-infected A. albopictus (C6/36) cells.

Intact viroplasmic centers were isolated from Estigmene acres cells infected with Tipula iridescent virus (TIV) by homogenization, followed by differential and discontinuous sucrose gradient centrifugation. Labeling of in situ and isolated viral assembly sites by two monoclonal antibodies raised against lymphocyte nuclear matrix proteins indicated a possible involvement of highly conserved nuclear proteins in the assembly and maturation of virions, as well as in maintaining the integrity of membrane-free viroplasmic centres. Electron microscopy and immunofluorescence of intact and fractionated E. acrea cells at different times postinfection showed no evidence of cytoskeleton involvement in the formation and maintenance of TIV viroplasmic centers.

Purified Tipula iridescent virus (TIV) from infected Galleria mellonella larvae was shown to consist of four DNA components: L (> 150 kilobase pairs (kbp)), S1 (10.8 kbp), S2 (~6 kbp), and S3 (~3.5 kbp). Analysis of the DNA from sucrose gradient fractionated TIV revealed that the fractions corresponding to partially filled virions have less of L relative to S1. All S components are related to L. S1 and S2 are the most similar, while S3 is a subset of S2. Three recombinant plasmids were isolated from a TIV DNA bank constructed in the BamHI site of pBR322. Two plasmids, pTB19-76 and pTB18-110, are related to S1 and S2 and they contain viral sequences that are repeated in the TIV genome. The third plasmid pTBR1-10 is related to S3. At least part of the variation in TIV genome size (174–246 kbp) obtained by summation of restriction endonuclease derived DNA fragments can be accounted for by the presence of the S components and sequence repetition.

Using light microscope autoradiography and electron microscopy we studied the effect of juvenile hormone III (JHIII) and β-ecdysone insect molting hormone (β-ecd) on the replication of Tipula iridescent virus (TIV) in suspension cultured cells of Estigmene acrea. JHIII at a concentration of 87.5 μg/ml completely inhibited viral DNA synthesis, but upon removal of JHIII, [3H]thymidine was incorporated into the cytoplasm as detected by autoradiography and virions in developmental stages from the same cell samples were-readily seen by electron microscopy. β-ecd at a concentration of 17.5 μg/ml, unlike JHIII, permitted viral DNA synthesis in the presence of the hormone although at a reduced level when compared to TIV-infected cells. But the presence of β-ecd seemed to prevent capsid formation, although islands similar in fine structure to those of viroplastic centers were seen by electron microscopy. Once β-ecd was removed from the medium, TIV-inoculated cells appeared to synthesize new virions in a normal pattern. Both hormones inhibited host cell DNA synthesis in noninfected cells.

The effect of sodium butyrate on Tipula iridescent virus (TIV) synthesis in suspension-cultured cells of Estigmene acrea was investigated. Sodium butyrate reduces viral-induced cell fusion but this is reversible with the removal of butyrate. At 7 mM sodium butyrate, TIV replicates in cells within 8 hr, but does not replicate in this time with 10–20 mm butyrate in the cell medium; cells so treated contain large vesicles with inoculum. Upon removal of the inhibitor, TIV replication appears normal, but large inoculum vesicles can still be found in the cytoplasm, and many infected cells have highly condensed chromatin in their nuclei. Sodium butyrate causes a lag of at least 2 hr in viral DNA synthesis as detected by [3H]thymidine incorporation into viroplasmic centres and at 7 mm butyrate viral DNA synthesis is reduced by 50–60%. In comparison, butyrate at 7 and 10 mm concentration does not inhibit host DNA synthesis, but at 15 and 20 mm, nuclear DNA synthesis is markedly reduced.

Infectious Tipula iridescent virus (TIV) inhibits thymidine incorporation in nuclei of infected cells as determined by light microscope autoradiography. In the electron microscope these nuclei have little condensed chromatin as compared to those of normal cells. Both noninfectious partially filled virions and empty capsids have a similar effect on nuclei as infectious TIV. When TIV is inactivated by uv, nuclei respond as though cells were inoculated with infectious TIV, but nuclei of cells inoculated with virus treated with β-propiolactone (BPL), or uv first, then BPL, incorporated thymidine and contained condensed chromatin clumps as normal cells.

Penetration of Tipula irisdescent virus (TIV) in suspension-cultured cells of Estigmene acrea occurred by viropexis within 1.5 hr postinoculation (pi), followed by the uncoating of the genome in pinocytotic vesicles. Viroplasmic centers appeared in the cytoplasm of the cells by 4 hr pi and increased in size and number until the majority of the cells displayed symptoms of viral synthesis. Assembly of progeny virions was restricted to isolated pockets of “loose matrix” within the viroplasm while viral DNA accumulated in surroundings areas of higher density viroplasm. Viral release occurred by exocytosis although cell lysis could not be excluded as a possible alternative. Cytopathic effects of TIV infection included cell fusion from 7–29 hr pi, alterations in nuclear morphology, and a rapid inhibition of host-cell macromolecular systhesis as measured by radio-isotope incorporation.

Electron microscopy of thin sections of salivary glands from wheat striate mosaic virus (WSMV)-infected leafhoppers, Endria inimica (Say), showed for the first time the presence of rhabdovirus particles in the leafhopper vector. These virus particles looked similar to those that have been observed in WSMV-infected wheat. The virions were found in the nuclei of infected cells both in well-defined intranuclear inclusions and in spaces between the inner and outer nuclear membranes. Bundles of particles were also seen in the cytoplasm close to infected nuclei. No particles were found in leafhoppers reared on virus-free wheat.

Callus cultures have been developed from leaf nodes of wheat (Triticum durum, Desf. Ramsey) infected with wheat striate mosaic virus (WSMV). This callus tissue consisted of discrete clumps of cells, with the youngest cells in the center and the oldest cells on the outside of the clump. Electron microscopic studies revealed numerous plasmodesmatal connections in young callus tissue; in addition the cytoplasm became less contrasted and large central vacuoles developed as the cells aged. WSMV particle were observed in abundance within both the nuclear envelope and the cytoplasm of the virus-infected callus cells. However, except for the presence of the virus, there did not appear to be any difference in the ultrastructure of WSMV-infected and healthy callus cells.

Viable cultures of protoplasts derived from wheat leaves (Triticum durum) were obtained. The protoplasts were isolated by enzymic degradation and kept viable in a defined medium supplemented with 1-glutamine, 2,4-D and kinetin.

Wheat striate mosaic virus was found to be an RNA virus composed of four major structural proteins having molecular weights of 25,000, 59,000, 92,000, and 145,000. Two of these proteins were isolated; one was identified as a glycoprotein (92,000 daltons) and the other was identified as a protein component (59,000 daltons) of the ribonucleoprotein.

Wheat striate mosaic virus (WSMV) systemically infects epidermal cells. The virus on rare occasions was found in guard cells. Although infection of subsidiary cells was common, guard cells bordering such subsidiary cells were generally free of virions. From a study of the relationship of plasmodesmata to mature cells of the wheat leaf epidermis, it appears that cytoplasmic connections are retained between subsidiary and adjacent epidermal cells after differentiation, but these connections are aborted between subsidiary and guard cells when the latter are fully differentiated. Thus, guard cell infection via plasmodesmata must occur at an early stage during cell differentiation.

The polypeptides in TIV and in TIV-infected hemocytes were studied by polyacrylamide gel electrophoresis (PAGE). TIV consists of 28 polypeptides ranging in molecular weight from 17,500 to 300,000. The lowest molecular weight polypeptide appears to be a lipoprotein, and none appear to be glycoproteins. Proteins, banding in the same area as TIV polypeptides, are seen in gels after PAGE of hemocytes from larvae 4 hr after viral injection. Also present in infected cells are 6 noncapsid viral polypetides. Infection by TIV is accompanied by an immediate depression of leucine incorporation into hemocyte and plasma proteins and a switch in synthesis from host-specific to virus-specific polypeptides.

Undulating filaments approximately 5 nm in diameter and of indeterminate length were observed in association with bacilliform virions of wheat striate mosaic virus (WSMV) in thin sections of infected wheat leaves. Virions were always at the periphery of the fibrillar regions and were found in the cytoplasm and nuclei of infected cells. The fibrillar areas and associated virus particles were not membrane bound. Two other morphologically different nuclear inclusions were observed which were restricted to virus-infected cells; their nature is unknown. The close association of the undulating filaments with virions is considered as evidence that the fibrils are the nucleoprotein component of WSMV.

Viral structural protein was localized in Tipula iridescent virus (TIV) infected hemocytes of Galleria mellonella larvae by fluorescent antibody and immunoferritin techniques. Viral and cell DNA were located by acridine orange staining.

HealthyVinca rosea plants, connected by dodder strands with spike-diseased sandal trees developed witches' broom symptoms. Electron microscopy of petioles of the infectedVinca plants revealed the presence of numerous mycoplasma-like bodies. Fresh dodder established on theVinca plants with witches' broom symptoms and trained onto healthy sandal trees, transmitted the spike disease back to the latter.

Electron microscopy of sections of leaves ( Sonchus oleraceus L.) infected with sowthistle yellow vein virus (SYVV) showed that the virus could infect parenchyma, developing xylem, and phloem cells. Virus was found in cells of leaves showing chlorosis and vein-clearing; none was seen in the adjacent green areas. Virus was localized predominantly in the nucleus and rarely in the cytoplasm. In either location, cell organelles were not interspersed with viral aggregates, although both polysomes and endoplasmic reticulum were normally at the periphery of cytoplasmic viral aggregates. In most cells areas of the perinuclear space were enlarged to accommodate aggregates of virus particles which were often invaginated into the nucleus.