Tipula Iridescent Virus Research Articles

Comparison of the major capsid protein genes, terminal redundancies, and DNA–DNA homologies of two New Zealand iridoviruses

Molecular comparisons were carried out on two iridoviruses isolated from endemic sympatric New Zealand pasture pests. These viruses, Costelytra zealandica iridescent virus (CzIV/IV16) and Wiseana iridescent virus (WIV/IV9), belong to the same virus genus but it is not known how related they are. The major capsid protein (MCP) gene from each virus was located, sequenced, and compared to the homologous gene from other iridoviruses. The MCP genes of WIV and CzIV were similar to each other (87.9% amino acid similarity) and to other iridovirus MCP genes. The MCP genes of both WIV and CzIV were most homologous to the MCP gene from Tipula iridescent virus (TIV/IV1), with amino acid similarities of 92.3 and 88.3% respectively. The genomes of WIV and CzIV were compared to other invertebrate iridoviruses using solution DNA–DNA hybridisations. Even after reducing the annealing stringency conditions hybridisation ratios never exceeded 10% indicating there is little sequence conservation between iridovirus genomes. Estimates of the size of terminal redundancies were also calculated for these viruses using pulsed-field agarose gel electrophoresis. These values ranged from 0 to 8%. These studies indicate that WIV and CzIV have distinct genomes and that the genus Iridovirus is comprised of a group of genetically diverse viruses.

Is the major capsid protein of iridoviruses a suitable target for the study of viral evolution?

Iridoviruses are large cytoplasmic DNA viruses that are specific for different insect or vertebrate hosts. The major structural component of the non-enveloped icosahedral virus particles is the major capsid protein (MCP) which appears to be highly conserved among members of the family Iridoviridae, Phycodnaviridae, and African swine fever virus. The amino acid sequences of the known MCPs were used in comparative analyses to elucidate the phylogenic relationships between different cytoplasmic DNA viruses including three insect iridoviruses (Tipula iridescent virus, Simulium iridescent virus, Chilo iridescent virus), seven vertebrate iridoviruses isolated either from fish (lymphocystis disease virus, rainbow trout virus, European catfish virus, doctor fish virus), amphibians (frog virus 3), or reptiles (turtle virus 3, turtle virus 5), one member of the family Phycodnaviridae (Paramecium bursaria Chlorella virus type 1), and African swine fever virus. These analyses revealed that the amino acid sequence of the MCP is a suitable target for the study of viral evolution since it contains highly conserved domains, but is sufficiently diverse to distinguish closely related iridovirus isolates. Furthermore the results suggest that a substantial revision of the taxonomy of iridoviruses based on molecular phylogeny is required.

Characterization of iridovirus IV1 polypeptides: mapping by surface labelling

A comprehensive index of IV1(Tipula iridescent virus, or TIV)-associated polypeptides has been established using enrichments of "empty" and "filled" virions that are considered to be maturation-phase-related. The mapping strategy which involved one- and two-dimensional polyacrylamide gel electrophoresis, silver staining, disulphide bond reduction and 125I iodination of putative surface proteins revealed 103 and 116 polypeptides for empty and filled capsids, respectively. These estimates could be reduced to < or = 70 by reclassing multicharged polypeptides of approximately identical masses as single entitles. At least 10 polypeptides from empty and filled virions were involved in intermolecular sulphhydryl linkages and another 11 species were identified as putative outer shell (surface) polypeptides. These data provide useful criteria for iridovirus classification and identification of candidate polypeptides involved in capsid formation and maturation.

Plaque assay and replication of Tipula iridescent virus in Spodoptera frugiperda ovarian cells

A plaque assay was developed for the study of Tipula iridescent virus (TIV) replication using a cell line derived from the fall army worm Spodoptera frugiperda (Sf9). Infection and plaque formation were monitored with time by phase contrast microscopy, video and fluorescent light microscopy. Structure of virions, viroplasmic centres and organelles of infected cells were examined by transmission electron microscopy (TEM). After 4 h postinfection, plaques were visibly detected within the cell monolayer by the presence of localized cell damage and production of numerous vesicular-like cytoplasmic structures. Quantitation of virions present per A260 unit of TIV preparation was determined by TEM. The number of visible plaques corresponded to virus concentration and 1 A260 produced approximately 10(5) plaques. DNA hybridization analysis revealed no gross differences in genomic DNA from TIV propagated in either Sf9 cells or wax moth Galleria mellonella larvae. These findings indicate that Sf9 is permissive for replication of TIV and superior by some parameters to other cell lines currently in use for the study of host cell/TIV interactions.

Identification of the gene encoding the major capsid protein of fish lymphocystis disease virus

The gene encoding the major capsid protein (MCP) of fish lymphocystis disease virus (flounder isolate; FLCDV-f) has been identified by PCR using oligonucleotide primers corresponding to different regions of the MCP of Tipula iridescent virus (TIV), iridescent virus 22 (IV22) and Chilo iridescent virus (CIV). DNA fragments of 0.4 kbp, 0.5 kbp and 0.27 kbp in size were amplified using oligonucleotide primers corresponding to amino acids (aa) 146 to 153 (primer 1) and 274 to 268 (primer 6), or aa 146 to 153 (primer 1) and 313 to 304 (primer 8), or aa 304 to 312 (primer 7) and 385 to 381 (primer 9) of the MCP of TIV, respectively. The PCR products were used as hybridization probes for screening the gene library of FLCDV-f. The MCP gene of FLCDV-f(1377 bp; 459 aa; 51.4K) was identified within the DNA sequence of the EcoRI FLCDV-f DNA fragment C (11.2 kbp; 0.611 to 0.718 map units). A high degree of aa sequence identity/similarity was detected between the MCP of FLCDV-f and TIV (50.3%/33.8%), IV22 (49.1%/34.2%). CIV (53%/29.5%) and African swine fever virus (16%/38.1%).

Identification of the gene encoding the major capsid protein of insect iridescent virus type 6 by polymerase chain reaction.

The gene encoding the major capsid protein of Chilo iridescent virus (CIV) has been identified by PCR using oligonucleotide primers corresponding to different regions of the major capsid proteins of Tipula iridescent virus (TIV) and iridescent virus 22 (IV22). A DNA fragment of 0.5 kbp was amplified using two oligonucleotide primers corresponding to the amino acid positions 146 to 153 and 304 to 313 of the major capsid protein of TIV, respectively. The radioactively labelled DNA fragment derived from PCR was hybridized to a CIV gene library. This analysis revealed that only the EcoRI CIV DNA fragment X [2.85 kbp; 0.589 to 0.603 viral map units (m.u.)] hybridized to the amplified DNA fragment. An RNA transcript of about 1.5 kb was identified when the PCR product was used as a hybridization probe. The same RNA transcript was detected when the EcoRI fragments X and Q (5.9 kbp; 0.603 to 0.631 viral m.u.) were used as probes. This indicates that the expected gene is located within map coordinates 0.589 to 0.631 and harbours part of the DNA sequences of fragments Q and X. The analysis of the DNA sequences of this particular region of the CIV genome revealed the presence of one open reding frame of 1401 bp. The DNA sequences of this region encode a protein of 467 amino acid residues with an M(r) of 51.4K. A high degree (64.7%) of amino acid sequence identity was detected between the major capsid protein of TIV and/or IV22 and the amino acid composition of the identified CIV protein.

A transfer membrane method for in situ detection and quantification of trehalase

A method for the detection and quantification of trehalase activity (EC 3.2.1.28) by immobilization to a membrane support has been developed. Protein samples partly enriched for porcine and Galleria mellonella wax moth larvae trehalase activities were fractionated by polyacrylamide gel electrophoresis, followed by electrophoretic transfer to PVDF membranes, and incubated in a solution containing trehalose (20 mg/ml), glucose oxidase (40 U/ml), phenazine methosulfate (0.06 mg/ml), and nitro blue tetrazolium (0.24 mg/ml) in 20 m m sodium phosphate buffer, pH 6.5. The intensity of the red-colored bands, developed directly on the membrane, was quantified using a computing, laser densitometer and shown to be linearly proportional to the original enzyme activity in extracts determined by liquid assay. The temperature inactivation profile of wax moth trehalase was measured. Alteration of the electrophoresis sample buffer composition further revealed the presence of putative trehalase isoforms in wax moth larval extracts whose relative levels of activity were altered during the course of starvation and infection with Tipula iridescent virus.

Semipermissive replication of Tipula iridescent virus in Aedes albopictus C6/36 cells

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 (>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.

Molecular cloning and characterization of a late Tipula iridescent virus gene

Virions of the cytoplasmic, icosahedral insect virus, Tipula iridescent virus (TIV), contain two major DNA components ( L, > 176 kb; and S1, 10.8 kb) and 25–30 proteins. We characterized a gene ( L96) whose 3.6-kb transcript is expressed late in the course of TIV infection of cultured of Estigmene acrea (salt marsh caterpillar, permissive host) and Aedes albopictus (mosquito, semipermissive host) cells. The L96 gene has an open reading frame of 867 codons, predicting a protein of 96 kDa with a pI of 10.9. The C terminus of the L96 protein is rich in hydrophobic amino acids and contains a small region of homology spanning a proteolytic cleavage site within two mammalian viral (GAG) polyproteins. Additional identity with H5 lysine-rich histones in the same region and with other DNA-binding proteins suggests that this protein may be involved in TIV structure. The lengths of the 5′- and 3′-untranslated regions of the L96 transcript were determined to be 21 nucleotides (nt) and 700 nt, respectively. Comparison of the TIV L96- and capsid-encoding genes, both of which are expressed late in infection, revealed that their 5′ and 3′ regions are generally rich in A and T residues, and that their 3′ ends encode at least one eukaryotic polyadenylation signal (AATAAA).

Molecular cloning, characterization, and expression of the Tipula iridescent virus capsid gene

The capsid protein is the major structural component of the icosahedral Tipula iridescent virus (TIV) that replicates in cytoplasmic inclusion bodies of insect cells. TIV capsid protein purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was digested with trypsin and fractionated by reverse-phase high-pressure liquid chromatography. A mixed oligonucleotide constructed from the amino acid sequence of a capsid tryptic peptide was used for the identification and cloning of the corresponding gene. The single-copy capsid gene, located on a 2.47-kilobase-pair HindIII TIV genomic fragment, codes for a 464-amino-acid protein (50,831 daltons) with a predicted pI of 6.34. Analysis of total RNA from infected Estigmene acrea cells indicated that the 1.8-kilobase capsid transcript was maximally produced between 14 and 24 h after infection. Transcript mapping by primer extension indicated that the RNA start site was in the A+T-rich TGCTACTAAT sequence, 19 nucleotides upstream from the first ATG codon of the capsid open reading frame. Expression of the TIV capsid protein in infected E. acrea cells was demonstrated by in vivo labeling of total proteins with [35S]methionine, using anti-capsid antiserum as the probe. Capsid protein was also expressed in Escherichia coli cells by using a pUC19 plasmid containing a lacZ-capsid gene fusion.

Association of nuclear matrix proteins with cytoplasmic assembly sites of Tipula iridescent virus

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.

DNA components of Tipula iridescent virus

Purified Tipula iridescent virus (TIV) from infected Galleria mellonella larvae was shown to consist of four DNA components: L (&gt; 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.

Comparison of microtubules and microfilaments in Tipula iridescent virus-infected and uninfected cells

Microfilaments and microtubules were detected in Estigmene acrea virus-infected cells using fluorescent immunochemistry and in sections by electron microscopy. Twelve hours following infection of cells with Tipula iridescent virus, large virus assembly sites developed in the cytoplasm. The majority of infected cells exhibit no detectable changes in the cytoskeleton during the initial stage of infection, when virus assembly sites are forming. Actin was localized either in cytoplasmic spikes or in patches at the cell surface. Microtubules were parallel to the long axis of elongate cells or randomly distributed in globular cells. Intermediate filaments were not detected using either immunofluorscent or electron microscopic techniques. In later stages of infection some cells exhibit a specific association between actin and the virus assembly site. The significance of this observation remains unclear since only a portion of the population exhibits this change. From this study, it does not appear that cytoskeletal elements are of importance in the formation or maintenance of the membrane-free cytoplasmic virus assembly sites.

The effect of two insect hormones on the replication of Tipula iridescent virus in Estigmene acrea cells in suspension culture

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.

A Tipula paludosa population with a high incidence of two pathogens

The incidence of lethal parasites in the larvae of a Tipula paludosa population was monitored for two seasons. The proportions of larvae infected with Tipula iridescent virus (TIV) and a tachinid insect were similar to those in previously studied populations, whereas the proportions of larvae infected with Tipula nuclear polyhedrosis virus (NPV) and a spore-forming bacterium (SFB) were higher. Conservative estimates of mortality due to these four agents were 10.7% in 1977–1978 and 7.7% in 1978–1979. The mean population density and the proportion of SFB-infected larvae were lower in 1978–1979 than in 1977–1978, while the proportion of NPV-infected larvae was higher. In 1979 the proportion of NPV-infected larvae was positively correlated with population density, which was highest in the wettest part of the study area. In both seasons the proportion of SFB-infected larvae was negatively correlated with population density. Larvae infected with the NPV or the SFB became pallid at an advanced stage of infection, but, although infected larvae were found throughout the larval period, pallid larvae were only found in the later part. It is suggested that larvae become infected in an early instar, then the infections slowly develop throughout the remainder of the larval period. Five larvae were found with mixed infections; four were infected with the SFB and NPV, while the fifth was infected with the SFB and TIV.

The effect of sodium butyrate on Tipula iridescent virus synthesis in insect suspension cell cultures

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 m M sodium butyrate, TIV replicates in cells within 8 hr, but does not replicate in this time with 10–20 m m 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 m m butyrate viral DNA synthesis is reduced by 50–60%. In comparison, butyrate at 7 and 10 m m concentration does not inhibit host DNA synthesis, but at 15 and 20 m m, nuclear DNA synthesis is markedly reduced.

The effect of hydroxyurea on DNA synthesis of Tipula iridescent virus and Estigmene acrea cells

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%.

Effect of virus inactivation on Tipula iridescent virus-cell relationships

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.

Localization of DNA phosphorus in Tipula iridescent virus by oxygen plasma microincineration. I. Electron microscopy of ash structures.

Tipula iridescent virus (TIV) is a relatively large particle containing about 15% DNA. As shown elsewhere by X-ray microanalysis (Thomas RS, Corlett M: J Histochem Cytochem 29:394, 1981), low-temperature oxygen plasma microincineration of the virus produces a stable phosphorus oxide ash representing this DNA nearly quantitatively. Transmission electron microscopy (TEM) of entire particles after plasma incineration shows the ash confined to the viral cores, confirming the previously known general location of the nucleic acid. Examination of ultrathin-sectioned virus crystals after plasma etching or ashing shows, on a still finer scale, that the DNA is probably confined to a shell structure within the core. A fine trace of ash from the capsid, seen in some preparations, may represent a phospholipid internal membrane known to be present. The possibilities of ash pattern artifacts are discussed. Heating experiments show that the ash patterns (and native virus particles) evaporate completely at high temperature, consistent with their presumed polyphosphoric acid composition. A heat-stable ash could be formed, however, when the viral DNA became accidentally stained with iron from the steel TEM grids used--a noteworthy artifact. The present work suggests some future possibilities of the plasma microincineration technique. In particular, the ability to see directly the fine distribution of mineral concentrations in ash patterns with the full resolution of TEM should be a powerful adjunct to increase effectively the sensitivity and resolution of X-ray microanalysis of mineral constituents in biological specimens.

Localization of DNA phosphorus in Tipula iridescent virus by oxygen plasma microincineration. II. Electron probe X-ray microanalysis of ash constituents.

Microincinerated, 1-micrometer sections of Tipula iridescent virus (TIV) crystals were analyzed quantitatively with a wave-length-dispersive electron microprobe to determine the chemical composition of high-resolution ash patterns previously obtained from ultrathin sections of the same specimen (Thomas RS: J Histochem Cytochem 29:379, 1981). Parallel analyses were performed on intact sections. In some cases, the same section and probe tracks were analyzed both before and after ashing. The principal elemental constituent of the ash was phosphorus, representing nearly all of the phosphorus found in the unashed sections. This confirmed the likely DNA origin of most of the ash. Other elements--sulfur, calcium, sodium, and perhaps carbon and nitrogen--found in relatively small concentrations in the ash, were partly due to either to incomplete ashing or to a particulate contaminant. Similar plasma ashing and analyses of sectioned model preparations of polymethacrylate containing dissolved triphenyl phosphate confirmed that phosphorus, by itself, could be retained as a stable ash. Analyses of polymethacrylate containing aluminum as well as phosphorus disclosed an unexpected artifact--aluminum inhibited the plasma ashing. These results suggested that the wavelength-dispersive probe, able to analyze for carbon and nitrogen as well as mineral elements, should be a generally useful tool in analyzing plasma microincineration phenomena, where macroscopic results do not apply. Relatively high beam intensity used throughout the probe analyses caused obvious damage to intact sections, particularly when they were mounted on thin-film supports. In contrast, the ash appeared quite stable. This suggested that plasma ashing of biological sections, converting them into mineralogical specimens, may be generally useful in probe analyses of mineral constituents, permitting greater sensitivity via higher beam currents, higher mineral concentrations, and lower backgrounds.