Infectivity and pathogenesis of iridescent virus type 22 in various insect hosts

Summary This paper reports the results of a series of laboratory experiments to determine the infectivity and pathogenesis of iridescent virus type 22 (IV 22) for six species of mosquitoes, phlebotomine sand flies and triatomid bugs. Following inoculation, IV 22 replicated in all of the species tested, without producing noticeable mortality within a 14 day observation period. Examination of the infected insects by immunofluorescence demonstrated large amounts of viral antigen in many different organs. Electron microscopy done on infected mosquitoes (Aedes aegypti) showed large numbers of virus particles within cells of the fat body, muscle tracheal and midgut epithelium. Virus replication in the mosquitoes was confined to host cell cytoplasm and was similar to that described in the natural blackfly (Simulium) host. Transovarial transmission of IV 22 could not be demonstraed inA. aegypti, and only a small percentage of mosquito larvae could be infected orally. Results of these experiments are compatible with observations of other iridescent viruses; IV 22 is highly infectious for a wide range of insects when introduced into their hemolymph, but it is not very infectious per os. These characteristics would appear to limit its value as a potential biocontrol agent for Diptera.     

The replication and titration of iridescent virus type 22 in Spodoptera frugiperda cells

A plaque assay for iridescent virus type 22 (from Simulium sp.) using Spodoptera frugiperda cells has been devised, and the kinetics of growth of the virus in this cell line have been determined. The virus particle/p.f.u. ratio was 75 +/- 8, and the p.f.u./TCID50 ratio was 0.56 +/- 0.11.     

Fatty acid profiles of invertebrate iridescent viruses

Summary Eight invertebrate iridescent viruses (IVs) from diverse host taxa were grown in a common lepidopteran host,Galleria mellonella. The lipid composition of purified virus was assessed by fatty acid methyl esterase (FAME) analysis using a gas-liquid chromatograph. IV fatty acid profiles were markedly different from those of the host tissues. The interrelationships among the IVs did not follow previous serological and genetic findings. We conclude that FAME analysis is not a useful technique for revealing phylogenetic relationships among these viruses.     

Comparison of the genomes of two sympatric iridescent viruses (types 9 and 16)

Summary A map of the sites in the genome ofCostelytra zealandica iridescent virus (CzIV), using the restriction enzymesBamHI,KpnI, andPstI, showed the genome size to be 170.2 kbp in length. It was found that the genome was cyclically permuted and that 39% of the genome of CzIV contained repetitive sequence elements. The genome was found to hybridize with the genome of another iridescent virus, type 9 (WIV), in DNA-DNA hybridization experiments. A region of the WIV DNA genome (23.4 kbp) did not hybridize with CzIV DNA and this region is similar in size to the total genomic size difference between CzIV and WIV (22.4 kbp). A unique repeat sequence from iridescent virus type 6 (CIV) was shown to be present in the genome of WIV but not that of CzIV. Finally, the positions of the major capsid protein genes, VP53 and VP52, in the restriction enzyme maps for type 16 and type 9 respectively were determined.     

Iridescent virus type 22 DNA

Summary Double stranded DNA extracted from iridescent virus type (IV22) was characterized by its buoyant density in CsCl, thermal denaturation profile and guanine plus cytosine content. The DNA was linear with a molecular weight of 130–143 × 10⁶ determined by reassociation kinetics, contour length measurements and restriction endonuclease analysis.With 3 Figures     

A cytological and immunological study of Tipula iridescent virus-infected Galleria mellonella larval hemocytes

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.Viral structural protein fluorescence was observed in the cytoplasm outside virus-induced viroplasms 1 day after virus inoculation. The viroplasm did not normally fluoresce until the second day. The intensity of fluorescence increased during the next 5 days. Specific ferritin tagging appeared on virus occurring singly in the cell, on virus infecting or leaving the cells, around groups of particles within the cytoplasm and throughout the viroplasm.Viral DNA was located exclusively in the viroplasm.Morphological and immunological data permitted evaluation of hypotheses of TIV assembly and a proposal is presented for the development and assembly of the virion in hemocytes.     

Production and characterization of the cores of the "R" strain of mosquito iridescent virus.

Cores of “R” strain of mosquito iridescent virus (RMIV) were produced in vitro by reacting intact virus with chymotrypsin. Isolation of the cores from undegraded virus and outer capsid protein was accomplished by density gradient and differential centrifugations. Negatively stained core particles had a diameter of 176.1 ± 6.0 nm when examined in the electron microscope. The density of the particles as measured in cesium chloride gradients was 1.33, and the s_20,w was 3126 as measured in the analytical centrifuge. The molecular weight of the cores was calculated to be 1.84 × 10⁹ daltons. Protein, DNA, and lipid analysis of the cores accounted for all but 48.0% of the protein of intact virus particles. SDS-polyacrylamide gel electrophoresis of the cores compared with that of intact virus showed that only a 55,000 molecular-weight protein was absent in the former. The cores did not infect larvae or an Aedes aegypti cell line, and serological comparisons of intact virus and cores showed the two did not share common surface antigens.     

Insect iridescent virus type 6 induced toxic degenerative hepatitis in mice

The toxic effect of insect iridescent virus type 6 — chilo iridescent virus — (CIV) was investigated using Balb/c mice (strain ByJ Ico and Kisslegg). The animals were inoculated with CIV intraperitoneally (1×10⁹ to 9.2×10¹¹ TCID₅₀/animal). The animals which were administered with 1×10¹¹ to 9×10¹¹ TCID₅₀ of CIV per animal, developed acute clinical illness and died during 18 to 80 h post infection. Histopathological and electronmicroscopic examinations of the liver tissues of those animals which died and/or were sacrificed when moribund showed acute degenerative hepatitis leading to death. No evidence for viral replication was found in the liver cells affected. A mortality rate between 21.1% and 100% was recorded for CIV, depending on the strain and number of mice used and the dose of virus administered. The toxic effect of CIV was eliminated or reduced extensively using heat denaturation or treatment of CIV with sodium dodecylsulphate or proteinase K. This indicates that the nature of the factor causing toxic degenerative cell damage is a protein.     

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.     

Physical mapping of the DNA genome of insect iridescent virus type 9 from Wiseana spp. larvae

A physical map for the DNA genome of insect iridescent virus type 9 isolated from Wiseana spp. larvae [Lepidoptera: Hepialidae] was constructed using the restriction enzymes BamHl, EcoRl, and Pstl. Viral DNA was cloned into the plasmids pBR328 and pUC8 using Escherichia coli strains HB101 and JM83, respectively. The physical map for BamHl, EcoRl, and Pstl was constructed by multiple enzyme analysis and Southern hybridization of cloned viral DNA. Statistical analysis of restriction data by computer-aided linear modeling supported the physical map produced and indicated a total genome size of 192.5 kb. Due to the cyclic permutation of iridescent virus genomes the map is presented in a circular form.     

A comparison of the DNA of the "R" and "T" strains of mosquito iridescent virus.

The buoyant density (1.7135 g/cm³), percentage GC (53.9%), and melting temperature (T_m = 76.4°) of the DNA of “regular” mosquito iridescent virus (RMIV) and of “turquoise” mosquito iridescent virus (TMIV) are similar although the molecular weights of the two DNAs are different; 243.3 × 10⁶ and 286.7 × 10⁶, respectively. Analyses show that RMIV contains two identical duplex DNA molecules which are about 15% smaller than the single duplex DNA molecule of TMIV. Reassociation studies show that RMIV and TMIV contain about 17 and 30% repetitious DNA, respectively. Homology studies show the two DNAs to be 100% homologous in their DNA nucleotide sequences. We conclude that the DNA of RMIV and TMIV contain identical sequences and that the portion of the TMIV-DNA molecule accounting for the higher molecular weight is composed of repeated sequences common to both strains.     

The DNA content of four small iridescent viruses: genome size, redundancy, and homology determined by renaturation kinetics

Fragmented DNA preparations from iridescent virus types 2, 6, 9, and 18 have been characterised for GC content and fragment size as a prelude to hybridisation studies. The genome size of the DNAs have been found to be 147 × 10⁶, 152 × 10⁶, 114 × 10⁶, and 114 × 10⁶ daltons for iridescent virus types 2, 6, 9, and 18, respectively, by studies of the kinetics of renaturation. Iridescent virus types 2 and 6 were found to contain a small proportion of DNA reannealing more rapidly than the bulk of the DNA, indicative of the presence of repeated sequences. In addition, the nucleotide sequence homology among the viruses has been investigated and found to range from 0 to 100%.     

Replication of Chilo iridescent virus in the cotton boll weevil, Anthonomus grandis, and development of an infectivity assay

Summary.  The boll weevil, Anthonomus grandis Boheman, is a devastating pest of cotton. Chemical pesticides are problematic due to relative lack of target specificity and resistance. Microbial pesticides may provide viable alternatives because of their narrow host range. Chilo iridescent virus (CIV) is the type species for genus Iridovirus, family Iridoviridae: large, icosahedral cytoplasmic viruses containing a double-stranded DNA genome. Earlier work suggested that CIV replicated in the boll weevil; however, efficiency or production of infectious virus was not established. We showed that CIV undergoes a productive cycle in A. grandis. CIV DNA levels in boll weevil pupae increased significantly from 0 to 3 days post infection. Moreover, virogenic stromata and complete virus particles were observed in the cytoplasm by 7 days. An endpoint dilution assay using viral DNA replication as indicator suggested a 10⁵-fold increase in infectious virus titer over 7 days. This is the first such demonstration in larval infections with genus Iridovirus. Our study establishes that CIV undergoes a productive cycle in the boll weevil and provides an important and useful model system for replication at the organismal level. These results have important implications for the potential of CIV and its components in boll weevil control. Received April 12, 2000 Accepted September 25, 2000     

Proteomic analysis of Chilo iridescent virus

In this first proteomic analysis of an invertebrate iridovirus, 46 viral proteins were detected in the virions of Chilo iridescent virus (CIV) based on the detection of 2 or more distinct peptides; an additional 8 proteins were found based on a single peptide. Thirty-six of the 54 identified proteins have homologs in another invertebrate and/or in one or more vertebrate iridoviruses. The genes for 5 of the identified proteins, 22L (putative helicase), 118L, 142R (putative RNaseIII), 274L (major capsid protein) and 295L, are shared by all iridoviruses for which the complete nucleotide sequence is known and may therefore be considered as iridovirus core genes. Three identified proteins have homologs only in ascoviruses. The remaining 15 identified proteins are so far unique to CIV. In addition to broadening our insight in the structure and assembly of CIV virions, this knowledge is pivotal to unravel the initial steps in the infection process.     

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.     

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.     

Three African isolates of small iridescent viruses: Type 21 from Heliothis armigera (Lepidoptera: Noctuidae), type 23 from Heteronychus arator (Coleoptera: Scarabaeidae), and type 28 from Lethocerus columbiae (Hemiptera Heteroptera: Belostomatidae)

Three small (130-nm) iridescent viruses have been found in invertebrates indigenous to Africa: Heliothis armigera, Heteronychus arator, and Lethocerus columbiae. Assessed by polyacrylamide-gel electrophoresis of the structural polypeptides and by serological tests (immunoneutralisation, immunoprecipitation, and complement fixation), the viruses represent related but not identical isolates.     

Ultrastructure of lymphocystis disease virus (LDV) as compared to frog virus 3 (FV3) and chilo iridescent virus (CIV): effects of enzymatic digestions and detergent degradations

Summary Ultrastructure of fish lymphocystis disease virus (LDV), the largest of all known icosahedral viruses, has been studied under electron microscopy using enzymatic digestions and detergent degradations. LDV structure appeared roughly the same as those of frog virus 3 (FV₃) and chilo iridescent virus (CIV), two other well known viruses of the familyIridoviridae, although the great flexibility of its capsid as observed on negatively stained and shadow cast particles, and its three electron dense layers visualized in ultrathin sections, differed from observations made with the two other viruses. Specific degradation of the virions with enzymes or detergents revealed that the composition of the three iridoviruses was very much alike. In fact, their capsid was composed of two layers as observed in negative staining: an external one, which was removed following digestion with proteinase K, and an internal one which could be digested with phospholipase A₂. Thus, the outermost layer is probably made of surface protein units, more or less tightly bound to each other, while the internal one would be a lipoprotein membrane. Consequently, these three iridoviruses appeared structurally related.     

Identification of a Bohle iridovirus thymidine kinase gene and demonstration of activity using vaccinia virus

Summary. In recent years interest in the family Iridoviridae has been renewed by the identification of a number of viruses, particularly from the genus Ranavirus, associated with disease in a range of poikilotherms. Ranaviruses have been isolated from amphibian, piscine and reptilian species. Here we describe an open reading frame (ORF) identified in the genome of Bohle iridovirus (BIV) which contains a nucleotide binding motif conserved within the thymidine kinase (TK) genes of iridoviruses from other genera (lymphocystis disease virus, LCDV, type species of the genus Lymphocystivirus; Chilo iridescent virus, CIV, type species of the genus Iridovirus). The ability of this putative gene to express a functional TK was confirmed by rescue of a TK negative mutant vaccinia virus in the presence of selective media, when expression was controlled by a vaccinia virus promoter. The sequence of the BIV TK was compared with the homologous sequences from epizootic haematopoietic necrosis virus (EHNV), a virus associated with disease in fish, from Wamena iridovirus (WIV) associated with systemic disease in green pythons, and from frog virus 3 (FV3) the ranavirus type species. Comparisons between these sequences and those available from other ranaviruses, other iridoviruses, other DNA viruses and cellular TKs are presented.     

Complete genome sequences of two begomoviruses infecting weeds in Venezuela

The complete sequences of isolates of two new bipartite begomoviruses (genus Begomovirus, family Geminiviridae) found infecting weeds in Venezuela are provided. The names proposed for these new begomoviruses are “Datura leaf distortion virus” (DLDV), isolated from a Datura stramonium L. (family Solanaceae) plant, and “Dalechampia chlorotic mosaic virus” (DCMV), isolated from infected Dalechampia sp. (family Euphorbiaceae) and Boerhavia diffusa L. (family Nyctaginaceae) plants. Phylogenetic analysis showed that these new begomoviruses segregated in two distinct clades of New World begomoviruses. To our knowledge, this is the first record of a begomovirus infecting Dalechampia sp. plants. Also, this is the first report of a begomovirus infecting Boerhavia spp. and Datura spp. in Venezuela.