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.     

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.     

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.     

Induction of apoptosis by iridovirus virion protein extract

Summary Chilo iridescent virus (CIV; IIV-6) is the type member of the genus Iridovirus (family Iridoviridae, large icosahedral cytoplasmic DNA viruses). CIV induces death and deformity in the cotton boll weevil, Anthonomus grandis, replicates productively in larvae of the cotton boll weevil, and significantly reduces laboratory populations of the cotton aphid, Aphis gossypii. CIV virion protein extract (CVPE) shuts down host protein synthesis in several insect cell lines and induces mortality in neonate boll weevil larvae. We report here that CVPE induces apoptosis in spruce budworm and boll weevil cell lines, as detected by blebbing, DNA fragmentation, and TUNEL assay. Tissue culture toxicity dose assays (TCTD₅₀) showed that spruce budworm cells were eight times more sensitive to CVPE than boll weevil cells. Pancaspase inhibitor suppressed apoptosis but had marginal effect on inhibition of host protein synthesis. Moreover, the CVPE dose for apoptosis was 1000-fold lower than the dose for shutdown of host synthesis. We also detected protein kinase activity in CVPE. Heating CVPE at 60 °C for 30 min destroyed all three activities. Our results suggest that one or more polypeptides in CIV induce apoptosis. This is the first study demonstrating apoptosis induction by a member of the genus Iridovirus and by virion extracts of a member of the family Iridoviridae.     

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.     

Insecticidal properties of phenols on Culex quinquefasciatus Say and Musca domestica L

Thirteen simple phenols and 8 phenolic acids were tested for toxicity to Culex quinquefasciatus larvae and Musca domestica adults. It was found that while the phenolic acids (except salicylic acid) showed little or no effect on acute toxicity, all the tested simple phenols caused mortality within 24 h after application. Lethal doses for acute toxicity of C. quinquefasciatus were successfully estimated for 12 substances using probit analysis. Thymol, carvacrol, 2-ethylphenol, and salicylaldehyde showed significantly the highest efficiency, for which the lethal doses LD₅₀ were estimated as 30, 36, 38, and 43 μg/ml, respectively. Lethal doses for acute toxicity of M. domestica adults were successfully estimated for ten substances. Thymol, carvacrol, and 2,6-dimethoxyphenol showed significantly the highest efficiency, for which the lethal doses LD₅₀ were estimated 53, 69, and 87 μg/fly, respectively.     

Purification of a toxic cysteine protease produced by pathogenic Aeromonas hydrophila isolated from rainbow trout

An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout (Oncorhynchus mykiss) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L ‐cysteine, iodoacetic acid, N ‐ethylamleimide, P‐chloromercuibenzene‐sulfonic acid and N‐α‐p‐tosyl‐1‐lysine‐chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS‐PAGE. In addition, the toxin was also completely inhibited by HgCl₂ but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl₂. Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD₅₀ values of 2.87 and 0.93 μg protein g^–1 fish body weight, respectively. The addition of L‐cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

Outcome of Influenza Infection: Effect of Site of Initial Infection and Heterotypic Immunity

An infection established throughout the total respiratory tract of mice with a highly lung adapted influenza virus (H0N1) led to death from viral pneumonia. The 50% lethal dose (LD₅₀) was approximately the same as the 50% infectious dose (ID₅₀). An infection with the same virus initiated in the nasal mucosa spread to the trachea and lungs over a 3- to 5-day period but was not lethal except at very high infecting doses. The LD₅₀ was 30,000 times the ID₅₀. Mice that had recovered from a prior infection with A/PC/73(H3N2) demonstrated enhanced recovery (heterotypic immunity) when challenged with A/PR/8/34(H0N1). Heterotypically immune mice infected while anesthetized with this potentially lethal virus stopped shedding virus from the nose, trachea, and lungs by day 7 and recovered. Heterotypically immune mice, infected awake, stopped shedding virus from the nose by day 5, and, in fact, the virus did not spread to the trachea or lungs. Thus, some of the variation in the severity of influenza infections may be explained by two factors: the site of initial infection and previous infection with heterotypic influenza virus.     

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.     

The nucleoprotein of Pichinde virus expressed by a vaccinia-Pichinde virus recombinant partially protects hamsters from lethal virus challenge

Summary Syrian hamsters, strain MHA/Lak, are susceptible to intraperitoneal infection with Pichinde virus and die from an overwhelming viremia. We have studied the ability of a vaccinia-Pichinde recombinant virus expressing amino acids 51-561 of the viral nucleoprotein (VVNP_51-561) to protect from lethal Pichinde virus infection. Priming with VVNP_51-561 significantly delayed mortality and increased final survival outcome after challenge with 2×10³ pfu of Pichinde virus. This protection was not complete compared to priming with Pichinde virus in the footpad, which was not lethal and provided 100% protection. At a higher challenge dose of Pichinde virus, 2×10⁴ pfu, immunization with VVNP_51-561 delayed mortality but did not increase final survival. The partial protection correlated with an early but not late reduction in infectious virus in serum, kidney and liver, and infectious centers in the spleen. Thus the immune response generated by VVNP_51-561 could initially control the infection, effectively reducing the virus inoculum. As the infection proceeded, virus replication could not be limited resulting in death in some hamsters. The partial protection did not appear to be mediated by anti-viral antibodies since these were not detected in the serum of VVNP_56-561-immunized hamsters. This finding appears to support the hypothesis that in many arenavirus infections cellular immunity is central to viral clearance and protection from reinfection.     

Serological relationships of an iridescent virus (type 25) recently isolated from Tipula sp. with two other iridescent viruses (types 2 and 22).

The isolation, purification, and serological properties of a 130-nm iridescent virus (IV), type 25, from Tipula sp. is reported. The polypeptide profile of this isolate was different from that of another small Dipterous iridescent virus-IV22 isolated from Simulium sp. However, these viruses were indistinguishable when compared by serum neutralization, gel immunodiffusion, complement fixation, tube immunoprecipitation, and immune electron microscopy. Iridescent virus type 2 isolated from Sericesthis pruinosa was shown to be distinct from IV22 and IV25 by all of these techniques.     

Predicting skin sensitization potential of organic compounds based on toxicity enhancement to Tetrahymena pyriformis,fathead minnow,and Daphnia magna

Skin sensitization is an important occupational health problem and immunotoxicity endpoint. Considering animal welfare and time and cost savings, many alternative approaches, such as those conducted in vitro, in silico, and in chemo, have been proposed and applied to predict skin sensitization of compounds. Toxicologically, sensitizers can elicit excess toxicity at greater levels than non-sensitizers due to their capacity to react with proteins/peptides. Based on this understanding, calculated toxicity enhancements (T_e) of 65 organic compounds from three in vitro bioassays, i.e. 48-hr ciliate (Tetrahymena pyriformis) growth inhibition, and both 96-hr fathead minnow and 48-hr Daphnia magna acute lethal toxicities, were employed to qualitatively and quantitatively predict skin sensitization potencies of the test agents. The sensitivity, specificity, and accuracy reaching 80% strongly suggested toxicity enhancement was an excellent parameter for predicting skin sensitization. Linear regressions of skin sensitization against toxicity enhancement were fitted for each bioassay, and they were improved after the sensitizers were categorized into different reaction mechanistic domains, which, in decreasing order of contribution from T_e to sensitization, were S_NAr?>?S_N1?>?MA. These results indicated that toxicity bioassays are useful tools and that T_e could be a useful parameter that might be applied to predict skin sensitization.     

Monovalent single-chain Fv fragments and bivalent miniantibodies bound to vesicular stomatitis virus protect against lethal infection

Several antibody-dependent mechanisms have been postulated to mediate neutralization of different animal viruses, including blocking of docking to receptors, induction of conformational changes in the virus coat, and Fc-dependent opsonization. We have studied the molecular requirements for antibody-mediated neutralization of vesicular stomatitis virus (VSV) in vitro and protection against lethal disease in vivo with a single-chain Fv fragment (scFv) and the corresponding bivalent miniantibody (scFv-dHLX) generated from a VSV-neutralizing monoclonal antibody. Both monovalent scFv and bivalent scFv-dHLX miniantibodies were able to neutralize VSV in vitro and to protect interferon-αβ receptor-deficient (IFN-αβR^−/−) mice against lethal disease after intravenous injection of 50 plaque-forming units (pfu) VSV pre-incubated with the scFv reagents. Similarly, severe-combined immunodeficient (SCID) mice infected with immune complexes of 10⁸ pfu VSV and bivalent scFv-dHLX were protected against lethal disease; however, mice infected with immune complexes of 10⁸ pfu VSV and monovalent scFv were not. Although repeated scFv-dHLX treatment reduced virus quantities in the blood, neither SCID nor IFN-αβR^−/− mice were protected against lethal disease after passive immunization and subsequent VSV infection. This was due to the short half-life of 17 min of scFv-dHLX in the circulation. These data demonstrate that neutralization of VSV and protection against lethal disease do not require Fc-mediated mechanisms and that cross-linking is not crucial for protection against physiologically relevant virus doses in vivo.     

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.     

Cellular pH and the ADH-induced hydrosmotic response in different ADH target epithelia

The hydrosmotic response elicited by oxytocin in the frog skin epithelium (Rana esculenta) was reversibly inhibited by 70% when the medium pH was reduced to 6.2 by CO₂ bubbling on the serosal side. On the contrary, the response to 8-bromo cyclic AMP (8 Br-CAMP) was not affected by medium acidification, even after corion removal. In other experiments intracellular pH was measured, employing the dimetyl-oxazolidine-dione distribution technique, in frog urinary bladder and the isolated frog skin epithelium. As previously observed in the case of oxytocin, 8 Br-CAMP increased intracellular pH in frog urinary bladder. Incubation with oxytocin also augmented the intracellular pH in the isolated frog skin epithelium but 8 Br-CAMP did not modify cell proton concentration in this tissue. From previous and present results it can be summarized that: 1) The intracellular alkalinization effect clicited by oxytocin addition and the inhibition in the hydrosmotic response induced by medium acidification were qualitatively similar in both tested target epithelia. 2) On the contrary, a post cyclic AMP step sensitive to changes in intracellular pH was not observed in frog skin, as is the case in frog urinary bladder. 3) The 8 Br-CAMP induced intracellular alkalinization effect was only observed in frog urinary bladder.     

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.     

Molecular Anatomy of Chilo Iridescent Virus Genome and the Evolution of Viral Genes

Chilo iridescent virus (CIV) or Insect iridescent virus 6 (IIV-6) is the type species of the genus iridovirus, a member of the Iridoviridae family. CIV is highly pathogenic for a variety of insect larvae and this implicates a possible use as a biological insecticide. CIV progeny and assembly occur in the cytoplasm of the infected cell and accumulate in the fatbody of the infected insects. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure and the amino acid sequences of different viral gene products. The elucidation of the coding capacity and strategy of CIV was the first step towards understanding the underlying mechanisms of viral infection, replication and virus-host interaction. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity of the CIV genome was determined by the analysis of the complete DNA nucleotide sequence consisting of 212,482bp that represent 468 open reading frames encoding for polypeptides ranging from 40 to 2432 amino acid residues. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs (468 ORFs) were non-overlapping. The identification of several putative viral gene products including a DNA ligase and a viral antibiotic peptide is a powerful tool for the investigation of the phylogenetic relatedness of this evolutionary and ecologically relevant eukaryotic virus.     

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