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The complete nucleotide sequence of a novel virus is presented here together with serological evidence that it belongs to Kashmir bee virus (KBV). Analysis reveals that KBV is a cricket paralysis-like virus (family Dicistroviridae: genus Cripavirus), with a non-structural polyprotein open reading frame in the 5' portion of the genome separated by an intergenic region from a structural polyprotein open reading frame in the 3' part of the genome. The genome also has a polyadenylated tail at the 3' terminus. KBV is one of several related viruses that also includes acute bee paralysis virus (ABPV). Although KBV and ABPV are about 70 % identical over the entire genome, there are considerable differences between them in significant areas of the genome, such as the 5' non-translated region (42 % nucleotide identity), between the helicase and 3C-protease domains of the non-structural polyprotein (57 % amino acid identity) and in a 90 aa stretch of the structural polyprotein (33 % amino acid identity). Phylogenetic analyses show that KBV and ABPV isolates fall into clearly separated clades with moderate evolutionary distance between them. Whether these genomic and evolutionary differences are sufficient to classify KBV and ABPV as separate species remains to be determined.

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Polydnaviruses are symbiotic viruses associated with some parasitic Hymenoptera that are vertically transmitted as proviruses within wasp genomes. To study this symbiotic association a gene encoding an abundant Campoletis sonorensis polydnavirus virion protein was characterized. This gene is not encapsidated but resides in the wasp genome where it is expressed only during virus replication. Immunolocalization studies detected the encoded 44-kDa protein only in oviduct tissue with ultrastructural studies detecting epitopes between or on virion envelopes. Expression and localization of the 44-kDa protein are consistent with its being a viral structural protein but localization of the gene only within the wasp genome is atypical, raising the possibility that this protein is adventitiously packaged during virion assembly. To address this possibility, quantitative dot blot and genomic Southern blot hybridizations were performed to determine whether the copy number of the p44 gene increased disproportionately during replication, as would be expected for a gene encoding a virion protein. The copy number of the p44 gene increases in tissues supporting virus replication but is unchanged in other tissues, suggesting that this gene is amplified in replicative cells. The data indicate that genes encoding polydnavirus virion proteins may be distributed between wasp and encapsidated viral genomes.

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A method for the activation and measurement of insect prophenol oxidase using nitrocellulose membrane is presented. Using this method we were able to conveniently activate both crude and purified prophenol oxidase from insects belonging to three different orders. This rapid method allows for prophenol oxidase activation, in the absence of a prophenol oxidase-activating system, and in the presence of high ionic strength, protease inhibitors, or chelator.

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Polymorphisms were readily detected in polydnavirus DNA extracted from several different species belonging to two different families of parasitic hymenoptera. Heterogeneity was observed as differences in electrophoretic profiles of genome segments, differences in the number of cross-hybridizing genome segments, and restriction fragment length polymorphisms; polymorphism was also detected at the level of an individual genome segment. Some implications drawn from these observations are discussed.

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Heterogeneity in polydnavirus DNA was exploited as a means of following the transmission of viral genomes in parasitoid populations. Parasitoid lines isogenic for viral DNA markers were established from both a braconid (Cotesia melanoscela) and an ichneumonid (Hyposoter fugitivus) species. In crossing experiments these markers routinely segregated in Mendelian (chromosomal) fashion, suggesting that the structure of polydnavirus genomes is probably determined by the integrated form of viral DNA, rather than by extrachromosomal molecules.

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Certain strains of the braconid parasitoid Cotesia melanoscela carry two different viruses within their ovaries, one of which (here designated CmV2) is apparently not a polydnavirus. Virus replication occurs in the ovarian calyx and in some other tissues of both male and female parasitoids; as yet, no replication has been observed in the testis, however. In addition, CmV2 is one of only two parasitoid viruses known to replicate in host insect larvae, and we not show that this virus is also capable of replicating in vitro; the virus is nevertheless nonpathogenic for gypsy moth larvae. The virus is not transmissible per os, either to host animals or to larvae of parasitoid strains lacking it. CmV2 is stably maintained within strains carrying it apparently by a vertical transmission mode involving the maternal line; transmission via the male germ line could not be demonstrated. While purification of the virus was not achieved, preliminary work allows us to suggest the genome consists of a single double-stranded DNA molecule of approximately 125 kb.

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Polydnaviruses are thought to replicate only in the ovaries of certain hymenopteran species. Nevertheless, in the present study, polydnaviral DNA was found to exist in males of the braconid parasitoid species Cotesia melanoscela and in both male and female non-ovarian tissue of an ichneumonid, Hyposoter fugitivus; preliminary results suggest that viral DNA may be present in an unintegrated form, but whether or not it is encapsidated is unknown. Using interstrain genetic crosses, we demonstrated that C. melanoscela males can apparently transmit at least some viral DNA to female progeny. We suggest that polydnavirus DNAs may be present in most if not all tissues of certain parasitoid species, and are probably maintained within parasitoid populations by vertical transmission through the germ line. In parallel experiments, manually injected eggs of the ichneumonid parasitoid (H. fugitivus) survived and hatched in Malacosoma americanum larvae in the apparent absence of exogenous polydnavirus; female parasitoids reared in this manner nevertheless carried virus in their ovaries. Experiments utilizing different strains of C. melanoscela also suggest that per os transmission of polydnaviruses (to parasitoid larvae) does not occur, despite the fact that inoculum viral DNA can be shown to persist for several days in the tissues of parasitized host larvae.

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Serological relationships among viruses isolated from the ovaries of some ichneumonid wasps have been examined by immunoblotting. An antiserum prepared against purified Hyposoter exiguae virus reacted with polypeptides of calyx fluids isolated from species belonging to several different ichneumonid genera. Significant cross-reactions, however, were confined to three genera and usually involved only two to four polypeptides.

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A virus that replicates in the ovary of a parasitoid wasp is injected into the parasitoid's host during oviposition. Successful development of th parasitoid egg within the host depends on the presence of th virus, which acts to suppress the host's immune response (encapsulation) toward the egg. This is an example of obligatory mutualism between a virus and a eukaryotic organism.

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A virus was found in the ovarial calyx tissue of Mesoleius tenthredinis, an ichneumonid parasitoid; the "infection" was present in all females thus far examined. Virions were morphologically similar to typical baculoviruses. Apparent uncoating of viral nucleocapsids at nuclear pores was observed, indicating the reinfection of the calyx epithelium may occur. Electron microscopy of DNA extracted from calyx tissue suggests that the viral genome may be circular, and of high molecular weight.

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Viruses are described from several genera of ichneumonid parasitoids. New morphologic categories have been observed, one of which is similar to typical baculoviruses. Calyx particles from several species were found to contain polydisperse DNA's. An electrophoretic method for screening individual field-collected wasps for the presence of such DNA's is reported. DNA profiles obtained by this procedure were sufficiently consistent, within any particular affected species, to suggest that they could provide useful taxonomic information.

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Virus-like particles have been isolated from the oviducts of a parasitoid wasp, Hyposoter exiguae (Hymenoptera: Ichneumonidae). Particles are readily purified by centrifugation on either Ficoll or sucrose gradients. Double-stranded circular DNA isolated from purified particles is heterodisperse in terms of molecular weight; none of the molecules are sufficiently large to code for the aggregate of structural proteins comprising the particles. Preliminary Southern blot hybridization data suggest that there is minimal sequence homology between the different size classes of DNA examined.

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A baculovirus present in the female reproductive tract of the parasitoid wasp Apanteles melanoscelus has been isolated and partially characterized. Viral DNA is double stranded, circular, and of highly variable molecular weight ranging from 2 x 10(6) to 25 x 10(6); the DNA is of homogeneous density at rho = 1.694 g/ml. Acrylamide gels resolve 18 polypeptide bands in the case of purified virions; four to five of these appear in a semipurified nucleocapsid preparation. The electrophoretic profiles obtained are compared with those of two other baculoviruses.

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Particles originating from the ovarial calyx epithelium of two different species of ichneumonid wasp are injected into host caterpillars during oviposition. At 1 3/4 h post oviposition, many calyx fluid particles are either associated with or have oenetrated through the basement membranes surrounding various tissues. Shortly thereafter, apparently intact particle nucleocapsids are observed in both the cytoplasm and nucleus of host cells. An unusual tubular protrusion of the viral envelope appears to be involved in either or both of penetration of basement membranes and entry of nucleocapsids into host cells.

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Nuclear inclusion bodies are found in the hemocytes of all tussock moth larvae parasitized by the braconid wasp Apanteles melanoscelus. These inclusion bodies represent the apparent site of replication of an unusual virus-like particle. Identical particles are observed in the nuclei of a small number of parasitoid calyx cells and are probably transmitted to host larvae during oviposition.

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101 pairs of human embryonic kidneys of 5-12 weeks' gestation were maintained in whole organ culture by our previously described technique, with herpesvirus hominis types1 and 2 added to our regular media. One kidney of each pair served as a control and was exposed to identical culture medium without virus. Cultures were maintained for 24-120 h to study the time sequence of viral infectivity. Organs were then examined for the presence of virus by electron microscopy and histochemical staining. Histological studies of virus-infected kidneys showed either (1) complete organ death or (2) virus localization in undifferentiated cells, plus disorganization of architecture in differentiated areas. Control organs showed normal organization.

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Virus-like particles have been found in specific regions of the reproductive tracts of three different braconid wasps, all parasitoids of the tobacco budworm, Heliothis virescens. The particles are nuclear in origin, and Feulgen cytochemistry of particulate fluid in the calyx and oviducts of one species has revealed the presence of DNA. On the basis of apparent structural homologies, it is suggested that the parasitoid particles are related to baculoviruses.

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