Iridovirus Type Research Articles

Genomic and Proteomic Analysis of Invertebrate Iridovirus Type 9

Iridoviruses (IV) are nuclear cytoplasmic large DNA viruses that are receiving increasing attention as sublethal pathogens of a range of insects. Invertebrate iridovirus type 9 (IIV-9; Wiseana iridovirus) is a member of the major phylogenetic group of iridoviruses for which there is very limited genomic and proteomic information. The genome is 205,791 bp, has a G+C content of 31%, and contains 191 predicted genes, with approximately 20% of its repeat sequences being located predominantly within coding regions. The repeated sequences include 11 proteins with helix-turn-helix motifs and genes encoding related tandem repeat amino acid sequences. Of the 191 proteins encoded by IIV-9, 108 are most closely related to orthologs in IIV-3 (Chloriridovirus genus), and 114 of the 126 IIV-3 genes have orthologs in IIV-9. In contrast, only 97 of 211 IIV-6 genes have orthologs in IIV-9. There is almost no conservation of gene order between IIV-3, IIV-6, and IIV-9. Phylogenetic analysis using a concatenated sequence of 26 core IV genes confirms that IIV-3 is more closely related to IIV-9 than to IIV-6, despite being from a different genus of the Iridoviridae. An interaction between IIV and small RNA regulatory systems is supported by the prediction of seven putative microRNA (miRNA) sequences combined with XRN exonuclease, RNase III, and double-stranded RNA binding activities encoded on the genome. Proteomic analysis of IIV-9 identified 64 proteins in the virus particle and, when combined with infected cell analysis, confirmed the expression of 94 viral proteins. This study provides the first full-genome and consequent proteomic analysis of group II IIV.

Identification of viral antigenic determinants by monoclonal antibodies directed against Chilo Iridescent Virus (Iridovirus type 6)

Monoclonal antibodies (McAbs) obtained against Iridovirus type 6 (CIV) were characterized by Western blotting and/or immunoprecipitation. Seven McAbs were found to be strongly reactive with viral polypeptides of molecular weights 16 K and 18 K by Western blotting. Two McAbs were directed against a complex composed of 30 K, 50 K, and 100 K polypeptides, but failed to react with either of these free polypeptides. This finding could explain the faint reactivity of these McAbs in Western blotting and immunoprecipitation. The reactivity of the other McAbs with their antigenic determinants is also discussed.

Phosphorylation of a ribosomal protein in invertebrate cells infected with ultraviolet-inactivated iridovirus type 6

Summary Chilo iridescent virus (CIV) infection induces a rapid shut-off of macromolecular synthesis in insect host cells. We have previously shown that CIV infection induced the appearance of new phosphoproteins in permissive cells of Choristoneura fumiferana (Cf-124) [15]. Among them, phosphorylation of a 31 Kd ribosomal protein (very similar to the S6 protein) was observed 3 h after infection of Cf-124 cells with UV-inactivated CIV. However, such phosphorylation could not be detected in cells infected native CIV in the same conditions. As previously postulated [15], these results could be interpreted either as an absence of phosphorylation of this ribosomal protein, or as a dephosphorylation step occurring very rapidly during the early stages of the replication cycle, in cells infected with native CIV, as reported for the infection of BHK21 cells by vaccinia virus [1].

Monoclonal antibodies against Chilo iridescent virus (iridovirus type 6). Brief report.

Mouse hybridomas producing antibodies against proteins of Chilo Iridescent Virus were established by fusions of X 63-Ag 8-653 myeloma cells with spleen cells from balb/c mice immunized with purified disrupted viral particles. Forty monoclonal antibodies to CIV proteins have been characterized. By ELISA, 4 categories of monoclonal antibodies were defined according to their ability to react with some selected viral protein fractions used as antigen. In preliminary studies, the specificity of 2 monoclonal antibodies for some viral polypeptides was determined by immunoblotting.

Modifications in the phosphorylation of ribosomal proteins and ribosome-associated proteins in invertebrate cells infected with iridovirus type 6

Summary Chilo iridescent virus (CIV) infection results in an increase in the phosphorylation of proteins. Early after infection, we observed three major new phosphoproteins in the ribosome salt-wash fraction of infected cells. These phosphorylations were not observed with virus which had been highly irradiated by UV irradiation. We therefore conclude that expression of the viral genome is required for these events. An increase in phosphorylation of ribosomal proteins was observed only when permissive cells were infected with UV-irradiated CIV, suggesting a host cell response rather than an expression of the viral genome.

Chilo suppressalis iridescent virus induced antigens in insect cells and tissues: Localization and kinetics of appearance

Summary The viral particle structural antigens of Chilo iridescent virus (CIV) (iridovirus type 6), an entomopathogen of Chilo suppressalis Walker (Lepidoptera: Pyralidae), were detected by the immunoperoxidase technique in permissive systems: Aedes aegypti, Spodoptera frugiperda and Choristoneura fumiferana cell lines and Galleria mellonella larvae. These antigens were first observed at 10–24 h post-infection in each permissive system. The percentage of infected cells was greater in vivo than in vitro and increased with higher temperatures of incubation (18 to 28°C). The staining was localized in the cell cytoplasm. As shown in electron microscopy, viroplasm and viral particles were specifically stained. Very shortly after in vitro infection (5–6 h) of A. aegypti, S. frugiperda and C. fumiferana cells, some early antigens were detected in the cytoplasmic area near the cellular membrane or within it. Detection was performed by the immunoperoxidase technique with sera raised against a crude extract of infected larvae of G. mellonella. Controls were always negative. The immunoperoxidase technique indicates that CIV-induced antigens appear sooner than previously described in the literature and that the amount of antigens increases with temperature. Moreover, evidence was obtained for the presence of one or more thus far unknown early antigens.

Isolation of polysomes from permissive and non-permissive invertebrate cell lines infected with chilo iridescent virus.

Chilo Iridescent virus (Iridovirus type 6 or CIV) infection results in a disaggregation of the heavy polyribosomes both in permissive and non-permissive invertebrate cell lines. The integrity of the viral genome is not involved in this event, as shown by the polysome absorbance profile on sucrose gradient, prepared from cells infected with UV treated virus. Heavy polysomes reappeared in permissive infected cells during the viral replication cycle.

Structural and thermodynamic investigation of the Chilo iridescent virus (Iridovirus type 6)

Structural features and thermodynamic parameters of the complete Chilo iridescent virus (Iridovirus type 6) and its constituents, isolated from the larvae of Galleria mellonella, were evaluated by means of UV spectroscopy and microcalorimetry. It can be demonstrated that the viral DNA is attached to the coat protein in a chromatin-like fashion, which is preserved after disruption of the virus by low temperature or partial digestion of the coat protein with proteinase K. At elevated temperature however the viral DNA is denaturated irreversibly. The coat protein appears to remain in its native state during the DNA transition and its own thermal denaturation profile shows its independence from the DNA denaturation.

Alkaline protease activity associated with iridescent virus type 6

An alkaline protease activity has been found to be associated with Iridovirus type 6. The enzyme showed a pH optimum of 10–10.5 with azocoll and hemoglobin but was essentially inactive on casein. From inhibitor studies the enzyme behaved like a serine protease. An Mr value of about 11500 was determined by SDS-PAGE.

Effect of infection with iridovirus type 6 on invertebrate cell phospholipids

Summary Iridovirus type 6 induced a modification in the phospholipids of invertebrate cells early in the infection cycle. This was shown by following the incorporation of radioactive phosphorus into the whole-cell phospholipids and analysing the percentages of the different phospholipid components after viral infection. We compared the results obtained on different permissive and non-permissive invertebrate cell lines. These results showed, with the whole-cell cultures used, that the viral infection led to a decrease in the phosphorus incorporation into the cell phospholipids and to a stimulation of the cell phosphatidylcholine synthesis.

Crossed immunoelectrophoretic characterization of Chilo iridescent virus surface antigens.

The surface antigens of Iridovirus type 6 (CIV) were characterized by crossed immunoelectrophoresis. Using different solubilization techniques, up to seven antigenic determinants corresponding to four viral structural polypeptides were identified.

Inhibition of macromolecular synthesis in cells infected with an invertebrate virus (iridovirus type 6 or CIV).

Chilo Iridescent Virus (CIV), an invertebrate virus, rapidly inhibits cellular RNA, DNA and protein synthesis in permissive and non permissive vertebrate and invertebrate cell lines. The integrity of the viral genome is not required for inhibitory expression, since viral proteins solubilized from CIV by freezing and treatment with EDTA exhibit inhibitory properties similar to those of intact virions.

Study of the structural polypeptides of Chilo suppressalis iridescent virus (Iridovirus type 6)

We report a procedure for the purification of Chilo iridescent virus (Iridovirus type 6), an evaluation of the purification procedure, and the results of analyses of the virion proteins by acrylamide gel electrophoresis. Purity was evaluated in three ways, i.e., by analysis of purified virions from artificial mixtures of infected and labeled uninfected larvae, electrophoresis at neutral pH, and electron-microscopic examination. Analysis of the polypeptides of purified CIV gave the following results: (i) after solubilization with SDS-B-mercaptoethanol, 16 polypeptides could be resolved in Coomassie brillant blue-stained electrophoretograms with molecular weights ranging from 18,000 to 115,000; (ii) after solubilization with SDS-urea, 26 polypeptides could be resolved with molecular weights ranging from 10,000 to 230,000 daltons.