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Dynamics of deletion genotypes in an experimental insect virus population.

Defective viruses, that are deficient in certain essential genes, are maintained in the population by trans-complementation, exploiting the gene products of complete genotypes in co-infected cells. This process becomes prevalent only when cells are frequently infected by several virus particles, and only then will the fitness of defective viruses be subjected to frequency-dependent selection. Deletion variants that are not infectious per os are present in a multicapsid nucleopolyhedrovirus (SfMNPV, Baculoviridae) that infects the fall army worm, Spodoptera frugiperda. These variants enhance the pathogenicity and, therefore, the likelihood of transmission of the virus when co-infecting cells with complete genotypes, resulting in occlusion bodies (OBs) that may contain both genotypes co-occluded. Mixtures of complete (B) and defective (C) variants in ratios of 90% B+10% C, 50% B+50% C and 10% B+90% C were used to inoculate by injection S. frugiperda larvae. Viral OBs extracted from diseased insects were subjected to four or five successive rounds of per os infection. Following successive passages, genotype frequencies in all three experimental populations converged to a single equilibrium frequency comprising approximately 20% of deletion genotype C and approximately 80% of complete genotype B. This mirrors the relative proportions of deletion (22%) and complete (78%) genotypes observed in the wild-type SfMNPV population. The pathogenicity of experimental populations at the final passage was not significantly different from that of the wild-type isolate. In contrast, OBs of all genotype mixtures were significantly more pathogenic than OBs of genotype B alone. A population genetics model, in which virus populations were assigned linear frequency-dependent transmissibility values, was in remarkably close agreement to empirical data. Clearly, non-infectious deletion variants can profoundly affect the likelihood of transmission and the genetic structure and stability of virus populations.

Animals↗

Comparative effects of a genetically engineered insect virus and a growth-regulating insecticide on microbial communities in aquatic microcosms.

The effects of a genetically engineered insect baculovirus on indigenous aquatic microbial communities were determined in closed, recirculating aquatic microcosms, and compared with the effects of a natural strain of the virus and of a growth-regulating insecticide, Dimilin. The recombinant virus was a nuclear polyhedrosis virus (NPV) of the spruce budworm (Choristoneura fumiferana (Cf)) with a lacZ marker gene inserted into the egt region of the CfNPV. The natural virus was Ireland strain CfNPV. Microbial measurement endpoints included decomposition activity (mass loss of organic material), respiration on two different substrates (O2 consumption), heterotrophic bacterial abundance (plate counts), and microbial community metabolic profiles (carbon source utilization patterns in Biolog GN microplates). Viral DNA of both the natural strain and the recombinant viruses, detected by polymerase chain reaction techniques, settled out of the microcosm water and accumulated on bottom substrates within 3 days of the microcosm inoculations. The viral DNA persisted in bottom substrates for the duration of the 21-day experimental period, although there was some evidence that the recombinant virus was less stable than the natural strain in particulate organic matter. No significant changes in microbial decomposition or respiration activity, bacterial abundance, or average metabolic responses were detected by a time trend analysis in microcosms inoculated with either the lacZ recombinant virus or the natural Ireland strain CfNPV. Significant effects on microbial decomposition and respiration activity were detected in microcosms treated with the growth-regulating insecticide at, and above, the expected environmental concentrations. Despite significant effects on microbial community functional attributes in Dimilin-treated microcosms, there were no detectable changes in community structure in terms of metabolic profiles or bacterial abundance.

DNA, Viral↗

Insect virus proteins (FALPE and p10) self-associate to form filaments in infected cells.

Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively. During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment. Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies. Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE). Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10. Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus. Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented. When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta moorei entomopoxvirus were observed. In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.

Amino Acid Sequence↗

Discovering new insect viruses: whitefly iridovirus (Homoptera: Aleyrodidae: Bemisia tabaci).

Adult whiteflies, Bemisia tabaci (Gennadius), collected from the field were screened for viral pathogens using a cell line from the silverleaf whitefly, B. tabaci, B biotype (syn. B. argentifolii). Homogenates from the field-collected whiteflies were applied to cell cultures and checked for cytopathic effects (CPE). Cells were observed to develop cytoplasmic inclusions and to have a change in morphology. Cells displaying CPE were observed using a transmission electron microscope and found to be infected with a virus. The virus particles had an icosahedral shape and an approximate size of 120-130 nm. The virus was observed in defined areas of the cytoplasm adjacent to the cell nucleus. Analysis using polymerase chain reaction, Southern blot hybridization, and DNA sequencing confirmed that the virus discovered infecting the whitefly cell cultures was an iridovirus. Sequence analysis showed that the amplimer (893 bp) had a 95% homology to the invertebrate iridescent virus type 6 major capsid protein gene. Discovery of new viruses of whiteflies may provide renewed interest in using pathogens in the development of innovative management strategies. This is the first report of an iridescent virus isolated from whiteflies, B. tabaci, collected from the field.

Animals↗

Insect virus: assays for viral replication and persistence in mammalian cells.

Viral pesticidal agents must be evaluated for potential health hazards prior to utilization. Assessment of the likelihood of replication in humans has included in vitro exposure of human cells to the potential pesticidal agent. Previous in vitro evaluation strategies have lacked positive controls. Thus, negative results, interpreted as no effect of the virus on human cells, could reflect basic deficiencies in the testing protocols. We designed a testing scheme for viral pesticides and used it to test the nuclear polyhedrosis virus of Autographa californica. Tests were aimed at evaluating potential replication or gene expression in primate cells. Parallel tests were run utilizing identical protocols with primate viruses known to produce the biological effect being evaluated. Thus protocols described were tested with positive viral controls.

Animals↗

Enveloped double-stranded DNA insect virus with novel structure and cytopathology.

An unusual type of virus has been isolated from larvae of the cabbage looper, Trichoplusia ni (Lepidoptera; Noctuidae). The virus infects a variety of tissues, including fat body, epidermis, and tracheal matrix, causing a chronic, fatal disease. Viral replication begins in the nucleus and is accompanied by invagination of the nuclear envelope and extensive nuclear and cellular hypertrophy. The nuclear envelope eventually ruptures and fragments, after which viral-induced membranes are assembled along planes through the cell and around its periphery. Subsequently, these membranes coalesce, partitioning most of the cell, including viroplasms and virions in various stages of assembly, among a cluster of vesicles. The vesicles dissociate and are liberated into the hemolymph where they accumulate in large numbers (>10(8) vesicles per ml), causing the blood to become opaque white. The virus has been isolated from T. ni and transmitted per os and by injection to T. ni and several other species of the family Noctuidae. The virions produced by this virus are large (ca. 130 x 400 nm), enveloped, and allantoid in shape with complex symmetry and contain apparently linear, double-stranded DNA of M(r) of approximately 1.00 x 10(8). The envelope contains subunits arranged in a hexagonal pattern that impart a distinctive reticular appearance to virions in negatively stained preparations. The unique structural and developmental properties of this virus indicate that it is a member of a group of enveloped, double-stranded DNA viruses not observed previously.

Journal Article↗

Timing of transmission and the evolution of virulence of an insect virus.

We used the nuclear polyhedrosis virus of the gypsy moth, Lymantria dispar, to investigate whether the timing of transmission influences the evolution of virulence. In theory, early transmission should favour rapid replication and increase virulence, while late transmission should favour slower replication and reduce virulence. We tested this prediction by subjecting one set of 10 virus lineages to early transmission (Early viruses) and another set to late transmission (Late viruses). Each lineage of virus underwent nine cycles of transmission. Virulence assays on these lineages indicated that viruses transmitted early were significantly more lethal than those transmitted late. Increased exploitation of the host appears to come at a cost, however. While Early viruses initially produced more progeny, Late viruses were ultimately more productive over the entire duration of the infection. These results illustrate fitness trade-offs associated with the evolution of virulence and indicate that milder viruses can obtain a numerical advantage when mild and harmful strains tend to infect separate hosts.

Analysis of Variance↗

[Interaction of vertebrate viruses and insect nuclear polyhedrosis viruses with transplantable diploid embryonal drosophila cells].

When the Drosophila cells were infected with the mixo- and arboviruses, in case of influenza A/WSN virus a rise in the titre and slight cytopathogenic effect with the subsequent decrease in the titre was observed. Since the decrease in the virus titer was not observed when actinomycin D was added, it was supposed that interferonlike inhibitor may be produced by the infected cells. Vacuolization and increase in the size of the infected cells were caused by all the nuclear polyhedrosis viruses tested. The number of the infected cells depended on the virus type and multiplicity of the infection.

Arboviruses↗

Genetically engineered rice resistant to rice stripe virus, an insect-transmitted virus.

The coat protein (CP) gene of rice stripe virus was introduced into two japonica varieties of rice by electroporation of protoplasts. The resultant transgenic plants expressed the CP at high levels (up to 0.5% of total soluble protein) and exhibited a significant level of resistance to virus infection. Plants derived from selfed progeny of the primary transformants also expressed the CP and showed viral resistance, indicating stable transmission of the CP gene and the trait of resistance to the next generation. Moreover, the virally encoded strip disease-specific protein was not detected in transgenic plants expressing CP 8 weeks after inoculation, indicating protection before viral multiplication. These studies demonstrated that CP-mediated resistance to virus infection can be extended to cereals and to the viruses transmitted by an insect vector (planthopper).

Base Sequence↗

Two kinds of BIR-containing protein - inhibitors of apoptosis, or required for mitosis.

The baculoviral IAP repeat (BIR) is a zinc-binding fold. Some BIR-containing proteins (BIRCs), including several from insect viruses, insects and vertebrates, are inhibitors of cell death and act by binding to active caspases. Their ability to do so can be antagonized by pro-apoptotic insect proteins such as Grim, HID and Reaper, or the mammalian protein Diablo/Smac. Members of one structurally distinct subgroup of BIR-containing proteins, which are present in yeasts and Caenorhabditis elegans as well as insects and vertebrates, do not act as caspase inhibitors; instead, they are required for chromosome segregation and cytokinesis, and act in concert with inner centromere protein (INCENP) homologues and aurora kinase homologues.

Animals↗

Studies on overwintering of bluetongue viruses in insects.

Bluetongue viruses (BTVs) are economically important arboviruses that affect sheep and cattle. The overwintering mechanism of BTVs in temperate climates has eluded researchers for many years. Many arboviruses overwinter in their invertebrate vectors. To test the hypothesis that BTVs overwinter in their vertically infected insect vectors, Culicoides sonorensis larvae were collected from long-term study sites in northern Colorado, USA, and assayed for the presence of BTV RNA by nested RT-PCR. Sequences from BTV RNA segment 7 were detected in 30 % (17/56) of pools composed of larvae and pupae collected in 1998 and in 10 % (31/319) of pools composed of adults reared from larvae collected in 1996. BTV was not isolated from the insects. Additionally, Culicoides cell-culture lines derived from material collected at one of the sites, or derived from insect samples collected during a BTV outbreak, contained BTV RNA segment 7. In contrast, segment 2 RNA was detected at half the rate of segment 7 RNA in the field-collected larvae and was only detected in the Culicoides cell lines with one of two primer sets. These data suggest that BTVs could overwinter in the insect vector and that there is reduced expression of the outer capsid genes during persistent infection.

Animals↗

Relationship of encephalomyocarditis virus to cricket paralysis virus of insects.

Cricket paralysis virus was shown to share common antigen(s) with encephalomyocarditis virus (Cardiovirus: Picornaviridae), a virus normally associated with mammals. These viruses must be regarded now as being related strains of one and the same virus. These results pose interesting questions for the taxonomy and ecology of small RNA viruses.

Animals↗