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Characterization of a novel satellite virus and a strain of Himetobi P virus (Dicistroviridae) from the brown planthopper, Nilaparvata lugens.

Two distinct spherical virus-like particles were purified from the brown planthopper, Nilaparvata lugens. One was a geographical isolate of Himetobi P virus (Cripavirus, Dicistroviridae). The other was 30 nm in diameter and contained positive-stranded RNA. The RNA was 1647 nucleotides in length and encoded only its own capsid protein, indicating that this particle is a satellite virus. Transmission tests showed that the satellite was transmitted vertically; however, its helper virus was unknown. We named this satellite Nilaparvata lugens commensal X virus (NLCXV).

Animals↗

Baculovirus replication: inhibition of Trichoplusia ni multiple nuclear polyhedrosis virus by [E]-5-(2-bromovinyl)-2'-deoxyuridine.

[E]-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) inhibits the replication of the baculovirus Trichoplusia ni multiple nucleocapsid nuclear polyhedrosis virus in Spodoptera frugiperda cells. Virus-specific DNA synthesis and late protein synthesis are suppressed by the drug. BVdU is phosphorylated by deoxythymidine (deoxycytidine) kinase present in both uninfected and virus-infected cells, and in its 5'-triphosphate form it inhibits DNA polymerase activity in virus-infected cells. The effect of the BVdU is not completely reversible. Phosphonoacetic acid, phosphonoformic acid and Acyclovir have no effect on baculovirus replication. Acyclovir fails to compete with deoxycytidine and thymidine as substrates for pyrimidine deoxynucleoside kinase in virus-infected and uninfected cells.

Animals↗

Deletion of the Autographa californica nucleopolyhedrovirus chitinase KDEL motif and in vitro and in vivo analysis of the modified virus.

Infection of insect larvae with Autographa californica nucleopolyhedrovirus (AcMNPV) results in the liquefaction of the host, a process involving the action of virus-encoded chitinase and cathepsin gene products. Chitinase is localized in the endoplasmic reticulum (ER) during infection because of the presence of a C-terminal ER retrieval motif (KDEL). In this study, the KDEL coding region was removed from the chitinase gene so that expression of the modified chitinase remained under the control of its own gene promoter, at its native locus. The deletion of KDEL resulted in the redistribution of chitinase within the cell during virus infection. Chitinase lacking the KDEL motif was detectable at the plasma membrane and was also evident in the culture medium of virus-infected cells from as early as 12 h post-infection (p.i.). Secretion of chitinase from the cell continued up to 72 h p.i., until cytolysis. The biological activity of the recombinant virus in Trichoplusia ni larvae was enhanced, with a significant reduction in the lethal dose and lethal time associated with infection. Furthermore, a reduction in feeding damage caused by infected larvae was observed compared to AcMNPV-infected individuals.

Amino Acid Motifs↗

Separation and structure of components of nuclear polyhedrosis virus of the silkworm.

Morphology of structural components of nuclear polyhedrosis virus (NPV) particles of the silkworm (Bombyx mori Linné) was studied by electron microscope using negative staining. NPV particles isolated from polyhedra could be separated into five structural components by centrifugation in sucrose density gradients. The lowest band (band I) was found to consist of thick rod-shaped particles (330 by 80 nm) with knobby surfaces and with occasional protrusion at one end. The second band from the bottom (band II) was shown to consist mainly of slender rod-shaped particles (360 by 60 nm), in which internal structures were visible as a dense mass. Regular striations were also seen on the surface of these particles. By treatment with mercaptoethanol, these particles were drastically damaged, and in some cases the internal substances were partially released, producing empty inner membranes of various degrees of disintegration. In bands III and IV, both empty spherical and empty rod-shaped membranes were present. Band III was rich in empty spherical membranes which were shown to be the outer membranes of thick rod-shaped particles. The empty rod-shaped membranes, the inner membranes, were mainly located in band IV and have cross striations on the surface. It is remarkable that the uppermost band (band V) consisted purely of small spherical particles, somewhat heterogeneous in size and shape (around 20 to 25 nm in diameter), indicating the particles to be the degradation product of the virus particles. Similar particles could also be observed within the empty inner membranes.

Animals↗

Altered phenotypic expression of ultraviolet-irradiated nuclear polyhedrosis virus.

UV (254 nm) irradiation of nuclear polyhedrosis virus results in formation of small plaques. The small-plaque effect is reversed by photoreactivation. Analysis of plaque formation kinetics indicates a slower formation and subsequent enlargement of plaques from UV-irradiated virus in comparison to non-irradiated virus. A delay in viral DNA synthesis is suggested as an explanation for the observed small-plaque effect.

Animals↗

A comparison of buoyant density and polypeptides of Drosophila P, C and A viruses.

On the basis of their buoyant densities in CsCl and their capsid polypeptides, three viruses isolated from Drosophila spp. which were originally described as serotypes, are now classified as distinct viruses. The biochemical properties of each virus suggest that it has several key features in common with the mammalian picornaviruses.

Capsid↗

Self-assembly of the JC virus major capsid protein, VP1, expressed in insect cells.

The major capsid protein of human polyomavirus JC virus, VP1, has been cloned into a baculovirus genome and expressed in insect cells. The VP1 protein was expressed in the cytoplasm and transported into the nucleus. It was then purified by a sucrose cushion and CsCI density gradient centrifugation to near homogeneity. Electron microscopy showed that isolated recombinant VP1 protein self-assembled into a capsid-like structure similar to the natural empty capsid. Both chelator (EDTA) and reducing agent (DTT) are required to disrupt the capsid structure into the pentameric capsomeres, as demonstrated by haemagglutination assay and electron microscopy. These results suggest that JC virus VP1 can be transported into the nucleus and self-assembled to form capsid-like particles without the involvement of the viral minor capsid proteins, VP2 and VP3. In addition, metal ions and disulphide bonds appear to be important in maintaining the integrity of the viral capsid structure.

Amino Acid Sequence↗

The plant virus Tomato Spotted Wilt Tospovirus activates the immune system of its main insect vector, Frankliniella occidentalis.

Tospoviruses have the ability to infect plants and their insect vectors. Tomato spotted wilt virus (TSWV), the type species in the Tospovirus genus, infects its most important insect vector, Frankliniella occidentalis, the western flower thrips (WFT). However, no detrimental effects on the life cycle or cytopathological changes have been reported in the WFT after TSWV infection, and relatively few viral particles can be observed even several days after infection. We hypothesized that TSWV infection triggers an immune response in the WFT. Using subtractive cDNA libraries to probe WFT DNA macroarrays, we found that the WFT's immune system is activated by TSWV infection. The activated genes included (i) those encoding antimicrobial peptides, such as defensin and cecropin; (ii) genes involved in pathogen recognition, such as those encoding lectins; (iii) those encoding receptors that activate the innate immune response, such as Toll-3; and (iv) those encoding members of signal transduction pathways activated by Toll-like receptors, such as JNK kinase. Transcriptional upregulation of these genes after TSWV infection was confirmed by Northern analysis, and the kinetics of the immune response was measured over time. Several of the detected genes were activated at the same time that viral replication was first detected by reverse transcription-PCR. To our knowledge, this is the first report of the activation of an insect vector immune response by a plant virus. The results may lead to a better understanding of insects' immune responses against viruses and may help in the future development of novel control strategies against plant viruses, as well as human and animal viruses transmitted by insect vectors.

Animals↗

Virulence variability of the Drosophila C virus and effects of the microparasite on demographic parameters of the host (Drosophila melanogaster).

We carried out experiments with the Drosophila C virus (DCV), a nonhereditary virus acting on demographic parameters of infected Drosophila host populations. It is well known that DCV increases mortality rate, decreases developmental time, and increases daily fecundity. As usual for Drosophila viruses, the DCV was multiplied in vivo. In this study we tested the hypothesis of virulence variability in DCV strains by isolating different stocks of the virus. The flies were tested for susceptibility to injection of such isolates and for virulence variability. Possible interactions between demographic parameters in three Drosophila host populations and injected isolates were studied under two egg densities (low and high). The hypothesis of virulence variability of DCV was supported by significant differences in mortality rates, depending on whether virus isolates were ingested or injected. When DCV was ingested, differences between host mortality rates were independent of the Drosophila host populations. Nevertheless, the developmental time was equally decreased by each virus isolate, independent of the host population. Moreover, the two viral stocks strongly increased the egg production of the flies. This experimental approach clearly showed that DCV could be considered a polymorphic virus. The phenotypic interactions between DCV and host flies varied according to parasite genotype.

Animals↗

Flock House virus: a nodavirus isolated from Costelytra zealandica (White) (Coleoptera: Scarabaeidae).

The physico-chemical properties of Flock House virus (FHV) were examined. FHV was shown to have a single-stranded RNA genome divided between two species of MW 1.1 and 0.46 million; the genome was infectious. FHV has a single, coat protein of 43,000 MW. The virus sediments at 142S and has a density of 1.351 g/ml in CsCl at pH 7. These properties indicate that FHV is a nodavirus but it is distinguishable from Nodamura virus and black beetle virus by differences in electrophoretic mobility, the size of the coat protein, particle density and stability in CsCl.

Coleoptera↗

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.

Animals↗

Triatoma patagonica (Hemiptera, Reduviidae), a new host for Triatoma virus.

Previous authors demonstrated that Triatoma virus (TrV) is able to infect several species of triatomines when injected with viral inoculum obtained from its original host, T. infestans. Both vertical (transovarian) and horizontal (faecal-oral) mechanisms of viral transmission were also described. In this paper we report the experimental TrV infection of a wild species from southern Argentina, T. patagonica. The inoculum consisted of clarified gut contents of infected T. infestans rubbed on the chicken skin whereupon T. patagonica individuals were fed. The results demonstrate that this is another potential host for the virus, and that the oral route is also effective for experimental interspecific infections.

Animals↗

Comparison of soluble and secreted forms of human parainfluenza virus type 3 glycoproteins expressed from mammalian and insect cells as subunit vaccines.

Human parainfluenza virus type 3 (PIV-3) is one of the leading causes of paediatric viral respiratory disease. The PIV-3 genome encodes two envelope glycoproteins, F and HN, which are the major targets for the host antibody response. We have expressed secreted forms of the F and HN proteins and a novel chimeric FHN glycoprotein in insect cells using recombinant baculovirus vectors and secreted forms of the F and FHN glycoproteins in stably transformed Chinese hamster ovary (CHO) cells. Comparison of the mammalian cell- and insect cell-expressed F and FHN proteins by SDS-PAGE showed that the CHO cell-expressed proteins are several kilodaltons larger in size than the baculovirus-produced proteins. A partial characterization of the oligosaccharide structures of the F and FHN proteins revealed that the size difference is due to the different oligosaccharide structures added to these proteins by the two cell lines. The F, HN and FHN proteins were immunoaffinity-purified from the culture medium of baculovirus-infected Sf9 cells and the F and FHN proteins were immunoaffinity-purified from the culture medium of CHO cells. A comparison of the immunogenicity and efficacy of the mammalian cell- and insect cell-produced FHN proteins was tested in cotton rats. The CHO cell- and baculovirus-produced FHN proteins were found to induce similar levels of PIV-3-specific ELISA-positive and neutralizing antibodies and both proteins provided near complete protection when animals were vaccinated with low doses of the FHN protein.

Animals↗

Functional studies on the p10 gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing a p10-beta-galactosidase fusion gene.

The beta-galactosidase gene (lacZ) of Escherichia coli was inserted in phase with the coding sequence of the Autographa californica nuclear polyhedrosis virus (AcMNPV) late-expressed Mr 10,000 (p10) gene. The fusion gene was inserted into the AcMNPV genome by cotransfection of a recombinant plasmid pAcR159Z, consisting of the EcoRI P fragment-containing pBR325-derived plasmid pAcR159 and the lacZ insert in the p10 gene, and wild-type AcMNPVDNA. Infection of Spodoptera frugiperda cells by the resulting recombinant AcMNPV/p10Z-2 showed high level expression of a p10-lacZ fusion protein, but no synthesis of p10. Therefore, the p10 gene is dispensable for virus replication and the p10 promoter is effective in driving the expression of foreign genes. Cells infected with AcMNPV/p10Z recombinants resembled those infected with wild-type AcMNPV in the amounts of polyhedrin synthesized and polyhedra formed, although p10 was absent. The nucleus and cytoplasm of AcMNPV/p10Z-2-infected cells lacked the fibrous structures that are associated with p10 in wild-type AcMNPV-infected cells. Instead, large granular structures were observed that were found by immunogold labelling to contain the lacZ gene product. The electron-dense 'spacers', thought to be precursors of the polyhedron membrane, were absent from cells infected by the recombinant virus and the polyhedra did not have a membrane. The recombinant AcMNPV/p10Z-2 was at least twice as virulent for second instar S. exigua larvae than was wild-type AcMNPV. The increased virulence of the recombinant is an important property for the control of insects.

Animals↗