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PubMed2002

Viral suppression of systemic silencing.

RNA silencing in plants is a form of antiviral defense that was originally discovered from the anomalous effects of transgenes. The process is associated with a systemic signal, presumed to be RNA, and is suppressed by plant virus-encoded proteins. One of these proteins, the 2b protein of cucumber mosaic virus, prevents systemic spread of the signal molecule but, curiously, is located in the nucleus of infected cells. The antiviral role of silencing might also apply in animals because a suppressor of silencing encoded by an insect virus was identified recently.

Animals↗
PubMed1994

Structures associated with the expression of rabies virus structural genes in insect cells.

When rabies virus structural genes were expressed in insect cells, major observed alterations were a high level of cytoplasmic vacuolization caused by the matrix protein M2 and the glycoprotein G. Ring-like structures, 16 nm in diameter, were observed in cell-free extracts from insect cells that expressed the N protein alone. Hexagonally shaped structures, 16-20 nm in diameter, and regular lattice aggregates of the same structures appeared on co-expression of N and M1 proteins. Co-expression of the four structural proteins led to the formation of cell surface blebs containing the structures corresponding to N and M1 proteins.

Animals↗
PubMed2005

Can plants use an entomopathogenic virus as a defense against herbivores?

It is by now well established that plants use various strategies to defend themselves against herbivores. Besides conventional weapons such as spines and stinging hairs and sophisticated chemical defenses, plants can also involve the enemies of the herbivores in their defense. It has been suggested that plants could even use entomopathogens as part of their defense strategies. In this paper, we show that Brassica oleraceae plants that are attacked by Myzus persicae aphids infected with an entomopathogenic parvovirus (M. persicae densovirus) transport the virus through the phloem locally and systematically. Moreover, healthy aphids that fed on the same leaf, but separated from infected aphids were infected via the plant. Hence, this is proof of the principle that plants can be vectors of an insect virus and can possibly use this virus as a defense against herbivores.

Animals↗
PubMed1986

Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector.

A baculovirus transfer vector was constructed using a cloned copy of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene. The ATG translation initiation codon and 32 bases of the polyhedrin mRNA 5' leader sequences were deleted and replaced with a cDNA clone of the A/PR/8/34 influenza virus haemagglutinin (HA) gene. The hybrid HA/polyhedrin gene was transferred to AcNPV and polyhedrin-negative recombinants identified which expressed HA activity. The insect cell-derived HA bound a monoclonal antibody specific for the HA gene in immunofluorescence tests and was shown to be plasma membrane-bound by haemadsorption assays. A 65 kDa protein was identified in infected cells by immunoprecipitation. The mRNA from recombinant-infected cells was analysed and revealed a major transcript of 2.95 kilobases consistent with transcription initiation and termination at the authentic polyhedrin sites. Other transcripts of different length were also detected so it was not possible to conclude which was responsible for producing the protein. The results demonstrated that AcNPV can be used to express plasma membrane-bound virus glycoproteins.

Animals↗
PubMed2002

Evolution of inhibitors of apoptosis in baculoviruses and their insect hosts.

The phylogenetic relationships of inhibitor of apoptosis (IAP) from insects and insect viruses were reconstructed and compared with the phylogeny of the viruses reconstructed on the basis of DNA polymerase species. The phylogeny supported the hypothesis that there were three IAP genes in the ancestor of the viral genus Nucleopolyhedrovirus (family Baculoviridae), but that there has been differential deletion of IAP genes in different lineages within this genus. An IAP gene from the granulovirus of the lepidopteran species Cydia pomonella (CpGV) was found to be a close relative of IAP genes from species of the insect order Lepidoptera, supporting the origin of this viral gene by capture of a host gene early in the evolution of Lepidoptera. The phylogeny supported the occurrence of least one other independent event of capture of an IAP gene by a virus and suggested the possibility of at least two other such events. Contrary to the prediction that host genes with viral homologues should experience an enhanced rate of amino acid replacement, no acceleration of evolutionary rate was detectable in these lepidopteran genes, which showed particularly low rates of non-synonymous nucleotide substitution in functionally important domains.

Animals↗
PubMed2002

Detailed characterization of polydnavirus immunoevasive proteins in an endoparasitoid wasp.

Polydnaviruses are a unique group of insect viruses in terms of their obligate and symbiotic associations with some parasitic wasps. The Cotesia kariyai polydnavirus (CkPDV) replicates only in ovarian calyx cells of C. kariyai female wasps and is injected into the wasp's host, the armyworm Pseudaletia separata, along with the eggs. A previous study indicated the possibility that one of the CkPDV surface proteins mediates immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes. This protein was named immunoevasive protein (IEP). The present studies substantially confirmed the previous observation by showing that an anti-IEP IgG neutralizes immunoevasive activity on the wasp eggs. Further, we isolated the IEP homologue (IEP-2) cDNA and IEP (IEP-1) cDNA, sequenced them and found that both are cysteine-rich proteins, each containing epidermal growth factor (EGF)-like repeats. IEP genes were not found to reside in the CkPDV genome, but in the wasp chromosomal DNA. IEPs are synthesized in the female reproductive tract and their expression was detected from 4 days after pupation, 1 day later than expression of the virus capsid proteins. In situ hybridization and immunocytochemistry indicated that the lateral oviduct cells of the reproductive tracts produce IEP-1/IEP-2 mRNAs and secrete the proteins into the oviduct. These data suggest that the expression pattern and localization of IEPs are different from other components of CkPDV virions.

Amino Acid Sequence↗
PubMed1985

The future of mosquito-borne diseases in the world.

The future of mosquito-borne diseases will depend on the improvements and implementation of chemotherapy and vaccination, as well as on biological and integrated control measures. Bacillus thuringiensis H-14, B. sphaericus, Lagenidium giganteum and other fungi are promising biological mosquito control agents. Other control measures include parasitoids, nematodes, larvivorous fish, Toxorhynchites mosquitoes, insect viruses, growth hormones, sex attractants, natural products, sanitation, and water management. Vector control should be combined with training of personnel and carried out on an international scale.

Animals↗
PubMed1998

Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex.

Hepatitis C virus (HCV) protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein into its constituent nonstructural proteins. The NS3/4A complex is thus an attractive target for antiviral therapy against HCV. We expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-terminal polyhistidine tag and purified the two proteins to homogeneity. Cleavage at the junction between HisNS3 and NS4A occurs during expression, producing a noncovalent complex between HisNS3 and NS4A with a subnanomolar dissociation constant. We purified the HisNS3/4A complex by detergent extraction of cell lysate and by metal chelate chromatography. We removed the His tag by thrombin cleavage and then further purified the complex by gel filtration. The purified NS3/4A complex is active in a protease assay using a synthetic peptide substrate derived from the NS5A-NS5B junction, with kcat/K(m) of 3700 (+/- 600) M-1 s-1, an order of magnitude above those previously reported for NS3 expressed by other strategies. This high protease activity implies that the full-length sequences of NS3 and NS4A are required for optimal activity of the NS3 protease domain. We examined the dependence of the NS3/4A protease activity on buffer conditions, temperature, and the presence of detergents. We find that, under most conditions, NS3 protease activity is dependent on the aggregation state of the NS3/4A complex. The monodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.

Adenosine Triphosphatases↗
PubMed1990

Isolations of African horse sickness virus from vector insects made during the 1988 epizootic in Spain.

This paper describes the first isolations of African horse sickness virus (AHSV) from insects in Spain. Seven isolations of AHSV serotype 4 were made; four from Culicoides imicola a known vector of the virus elsewhere, two from mixed pools of Culicoides species not including C. imicola and one from blood engorged mosquitoes. Three further isolations of AHSV serotype 4 were also made from horses kept adjacent to the insect collecting sites. This work presents the first definitive identification of the vectors of AHSV in Spain during the 1987, 88 and 89 epizootics. Suggestions are also made concerning the significance of these findings with regard to the epidemiology of African horse sickness in Spain.

African Horse Sickness↗
PubMed1999

Nucleotide sequence of a 5892 base pairs fragment of the LsMNPV genome and phylogenetic analysis of LsMNPV.

A 5892 bp fragment of Leucania separata multiple nuclear polyhederosis virus (LsMNPV) containing gp37, 39 k genes and another four ORFs was sequenced in this article. According to regulatory elements on 5' uncoding sequences, the ORF4 and ORF1 are probably two novel baculovirus genes, and the ORF4 probably also is a new early-late gene. The possible functions about GP37 and 39 K proteins were explored. The homology of GP37 protein among LsMNPV and 8 other insect virus was compared. The evolutional position of LsMNPV is also estimated based on the homology of gp37 genes. The structure and its homology of several genes in LsMNPV prove that LsMNPV is far related with other baculovirus and has an exceptional genome organization.

Amino Acid Sequence↗
PubMed1994

Mutation of either of two cysteine residues or deletion of the amino or carboxy terminus of nonstructural protein NS1 of bluetongue virus abrogates virus-specified tubule formation in insect cells.

Virus-specific tubules are characteristic of orbivirus infections and are likely to play an important role in virus morphogenesis. It has been shown that for bluetongue virus (BTV), the prototype orbivirus in the family Reoviridae, the virus-encoded NS1 protein forms tubules in insect cells when the BTV segment M6 gene is expressed by using a baculovirus vector. To understand the function of NS1 tubules and to identify the sequences involved in their polymerization, a series of mutant NS1 genes was generated and expressed in insect cell cultures by using baculovirus vectors. Three of the mutants were deletion mutants. One (AcNS1.dNT10) lacked 10 of the amino-terminal amino acids, and the other two mutants (AcNS1.dCT20 and AcNS1.dCT43) lacked 20 or 43 of the carboxy-terminal amino acids. In addition, site-directed mutants were constructed in which various single cysteines or pairs of cysteines were changed to serines. The ability of each mutant protein to form tubules was investigated. None of the deletion mutants formed tubules. The constructs in which the cysteines at amino acid positions 337 and/or 340 were replaced by serines (e.g., AcNS1.C337S,C340S) also did not form tubules. Instead, the NS1 protein of these and the deletion mutants made ribbon-like structures which formed large aggregates. Mutations involving six other cysteines (i.e., AcNS1.C37S,C43S,AcNS1.C462S,C465S, AcNS1.C104S, and AcNS1.C364S) produced tubules. The results show that both the amino and carboxy termini of the NS1 protein molecule and the cysteines at residues 337 and 340 are essential for tubule formation.

Animals↗
PubMed1969

Pathology and Development of the Grasshopper Inclusion Body Virus in Melanoplus sanguinipes.

Grasshoppers, Melanoplus sanguinipes (F.), infected with the grasshopper inclusion body virus (GIBV) showed a general torpor, took longer to develop, and had abnormally high rates of mortality. Infection was found only in the fat body, and developing viruses and inclusion bodies were observed in the nuclei and cytoplasm of infected cells. Although the size of the inclusion bodies in cells varied at different stages of infection, the inclusion bodies appeared to grow during the infection. Electron microscopic investigations of viral replication showed that at about 8 days after inoculation presumptive viral particles had developed as buds or protrusions from precursor granular masses; thereafter, these particles underwent internal differentiation and were incorporated into developing inclusion bodies. The GIBV was similar to insect viruses in the genus Vagoiavirus Weiser and to pox viruses, particularly vaccinia.

Journal Article↗
PubMed2002

Influence of the host plant on occluded virus production and lethal infectivity of a baculovirus.

Intra- and inter-specific effects of cotton, soybean, and clover on the time until death of Helicoverpa zea (Boddie) and Heliothis virescens (F.) larvae lethally infected with H. zea nucleopolyhedrovirus (HzSNPV) were evaluated in the laboratory. In the first test, on second instar only, the time until death of lethally infected larvae of both species differed with the plant tissues (vegetative or reproductive) and plant species. The total viral activity produced per larva in LC(50) units (occluded viral bodies (OBs) per larva/LC(50) in OBs/mm(2) of diet surface) was greater from H. virescens larvae fed vegetative than reproductive tissues of all host plants, but from H. zea virus production was greater only when fed vegetative tissue of soybean. In a second test that compared second and fourth instar H. virescens on cotton, total viral activity from larvae treated in both instars was greater when fed vegetative than reproductive tissues. Results of these tests suggest that the ability of host plants to influence baculovirus disease is more complex than previously believed. When examining the epizootic potential of a baculovirus, more attention must be given to the effects of the host plant on the insect-virus interactions.

Animals↗
PubMed1984

The effect of paraffin wax embedding procedures on the ability of the fluorescent antibody test to demonstrate bluetongue virus antigen in insect tissue.

The effect of the reagents used in paraffin wax embedding procedures on the fluorescent antibody technique was investigated with a view to using this test for virus location studies on paraffin wax sections of bluetongue virus infected Culicoides variipennis. Experiments were carried out using tissue smears rather than prepared sections, enabling the effect of individual reagents to be evaluated. Infected smears treated with acetone alone showed bright specific fluorescence, while treatment of infected smears with all other reagents, including low melting point wax and acetone-soluble ester wax, prior to immunofluorescence testing reduced the brightness of specific fluorescence or rendered the test completely ineffective.

Animals↗
PubMed2005

Amsacta moorei Entomopoxvirus inhibitor of apoptosis suppresses cell death by binding Grim and Hid.

Inhibitor of apoptosis (iap) genes have been identified in the genomes of two independent families of insect viruses, the Baculoviridae and the Entomopoxvirinae. In this report, we examined the functional attributes of the Amsacta moorei entomopoxvirus-encoded IAP protein (AMV-IAP). The binding specificity of the individual baculoviral IAP repeat (BIR) domains of AMV-IAP was investigated by using a random-peptide, phage display library, and sequences similar to the amino termini of proapoptotic Drosophila proteins in the Reaper/Hid/Grim family were identified. Furthermore, the BIR domains of AMV-IAP protein were demonstrated to bind the mammalian IAP inhibitor Smac through the AVPI tetrapeptide sequence, suggesting that the peptide binding pocket and groove found in the insect and mammalian IAPs is conserved in this viral protein. Interaction analysis implicated BIR1 as the high-affinity site for Grim, while BIR2 interacted more strongly with Hid. Both Grim and Hid were demonstrated to interact with AMV-IAP in vivo, and Grim- or Hid-induced cell death was suppressed when AMV-IAP was coexpressed.

Animals↗
PubMed1999

Vesicular stomatitis.

Vesicular stomatitis is a disease of livestock caused by some members of the Vesiculovirus genus (Family Rhabdoviridae), two of which are called 'vesicular stomatitis virus'. Clinical disease presents as severe vesiculation and/or ulceration of the tongue, oral tissues, feet, and teats, and results in substantial loss of productivity. Except for its appearance in horses, it is clinically indistinguishable from foot-and-mouth disease. Unlike foot-and-mouth disease, it is very infectious for man and can cause a temporarily debilitating disease. Vesicular stomatitis occurs seasonally every year in the southeastern USA, southern Mexico, throughout Central America and in northern South America, and emerges from tropical areas to cause sporadic epidemics in cooler climates during the summer months. Other Vesiculoviruses are endemic in India and Africa. Vesiculoviruses are arthropod-borne and it is possible they are actually well adapted insect viruses that incidentally infect mammals. Vesiculoviruses are relatively simple, having a linear, single stranded, negative sense RNA genome encased in a bullet-shaped virion made from only five proteins. Upon infection of cultured cells, viral products turn off cellular gene expression and seize the entire metabolic potential of the cell. They also depolymerize the cytoskeleton to cause rapid tissue destruction. Virus infection in animals provokes interferon and nitric oxide responses, which quickly control viral replication, and an antibody response that prevents further viral replication. Vesiculovirus genome replication is error-prone, resulting in viral progeny containing many variants. This allows rapid adaptation. Nevertheless, vesicular stomatitis virus genomic sequences appear relatively stable within single endemic areas, and vary progressively on a North-South axis in the Western Hemisphere. Numerous important fundamental discoveries in immunology and virology have come from recent studies of vesicular stomatitis virus. However, these discoveries have not led to a safe and fully effective vaccine for man or beast. In the absence of a vaccine, the continual increase in rapid intercontinental travel, the increase in numbers and concentration of susceptible animals, the plasticity of the viral genome, and the underappreciation of vesiculoviruses as veterinary and zoonotic pathogens by regulators and biomedical researchers, are combining with potentially explosive consequences.

Animals↗
PubMed2006

Detection and titration of bluetongue virus in Culicoides insect cell culture by an antigen-capture enzyme-linked immunosorbent assay.

Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. Virus is typically isolated and characterized by infection of susceptible vertebrate cells that undergo detectable and measurable cytopathic effects. Cell lines derived from C. sonorensis may be useful for virus isolation and studies to better understand BTV replication in the insect vector. However, their use is hampered because BTV infection does not produce significant cytopathic effects in these insect cell cultures. Detection of virus replication in these cells typically requires co-cultivation with susceptible vertebrate cell culture. This report describes the use of an antigen-capture enzyme-linked immunosorbent assay (Ag-Cap ELISA) for direct detection and titration of BTV in cultures of a Culicoides cell line. This assay should facilitate use of this cell line for virus isolation, titration and studies of BTV replication.

Animals↗