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PubMed1996

Handling small arbovirus vectors safely during biosafety level 3 containment: Culicoides variipennis sonorensis (Diptera:Ceratopogonidae) and exotic bluetongue viruses.

Equipment and procedures are described for biosafety level 3 (BL-3) containment work with small, zoophilic arthropods. BL-3 classified pathogens always must be manipulated in biological safety cabinets. Procedures, including physical barriers and handling methods, that prevent the escape of potentially virus-infected insects are discussed, and the use of a monitoring system for insect security is explained. The inability to recover escaped minute, flying insects poses a major difference from similar work with larger insects, such as mosquitoes. Methods were developed for the safe and secure handling of Culicoides variipennis sonorensis Wirth & Jones infected with exotic bluetongue viruses during BL-3 containment.

Animals↗
PubMed1994

Chilo iridescent virus encodes a putative helicase belonging to a distinct family within the "DEAD/H" superfamily: implications for the evolution of large DNA viruses.

The complete nucleotide sequence of the EcoRI DNA fragment M (7099 bp; 0.310-0.345 map units) of the genome of insect iridescent virus type 6--Chilo iridescent virus (CIV)--was determined. A 606 codon open reading frame located in this region encoded a protein (p69) related to a distinct family of putative DNA and/or RNA helicases belonging to the "DEAD/H" superfamily. Unique sequence signatures were derived that allowed selective retrieval of the putative helicases of the new family from amino acid sequence databases. The family includes yeast, Drosophila, mammalian, and bacterial proteins involved in transcription regulation and in repair of damaged DNA. It is hypothesized that p69 of CIV may be a DNA or RNA helicase possibly involved in viral transcription. A distant relationship was observed to exist between this family of helicases and another group of proteins that consists of putative helicases of poxviruses, African swine fever virus, and yeast mitochondrial plasmids. It is shown that p69 of CIV is much more closely related to cellular helicases than any of the other known viral helicases. Phylogenetic analysis suggested an independent origin for the p69 gene and the genes encoding other viral helicases.

Amino Acid Sequence↗
PubMed1997

Separate Expression and Characterization of the Heavy- and the Light-chain of A HBsAg Specific Murine-human Chimeric Antibody in Insect Cells.

Both the heavy- and light-chain of a murine-human chimeric antibody with specificity for hepatitis B virus surface antigen have been expressed separately in a baculovirus expression system. The heavy- and light-chain variable region (VH and VL) genes encoding the marine-monoclonal antibody OH3 were combined with human gamma3 and kappa constant region genes to construct the murine-human chimeric genes, respectively. The transfer vectors containing the chimeric genes were cotransfected into Sf9 cells with linearized virus DNA and recombinant viruses were isolated by dot hybridization, PCR and Southern blot analysis. The chimeric heavy- and light-chain was expressed respectively in the recombinant viruses-infected insect cells and the characterization by Western blot and competitive ELISA demonstrated that both of the expressed chimeric heavy- and light-chain have the specificity of HBsAg binding.

Journal Article↗
PubMed1981

N-Terminal polyhedrin sequences and occluded Baculovirus evolution.

A phylogenetic tree for occluded baculoviruses was constructed based on the N-terminal amino acid sequence of occlusion body proteins from six baculoviruses including three lepidopteran nuclear polyhedrosis viruses (NPVs), [two unicapsid (Bombyx mori and Orgyia pseudotsugata) and one multicapsid (Orgyia pseudotsugata)]; one granulosis virus (Pieris brassicae); and NPVs from a hymenopteran (Neodiprion sertifer) and a dipteran (Tipula paludosa). Amino acid sequence data for the B. mori NPV were from a report by Serebryani et al. (1977) and that for the O. pseudotsugata NPVs were reported previously by us (Rohrmann et al. 1979). The other N-terminal amino acid sequences are presented in this paper. The phylogenetic relationships determined based on the molecular evolution of polyhedrin were also investigated by antigenic comparisons of the proteins using a solid phase radioimmune assay. The results indicate that the lepidopteran NPVs are the most closely related of the above group of viruses and are related to these viruses in the following order: N. sertifer NPV, P. brassicae granulosis virus, and T. paludosa NPV. These data, in conjunction with Baculovirus distribution and evidence concerning insect phylogeny, suggest that the Baculovirus have an ancient association with insects and may havae evolved along with them.

Amino Acid Sequence↗
PubMed1988

Resistance of the 64K protein of budded Autographa californica nuclear polyhedrosis virus to functional inactivation by proteolysis.

The 64K surface protein of budded Autographa californica nuclear polyhedrosis virus (AcMNPV BV) is known to play a role in the functional entry of AcMNPV BV into Spodoptera frugiperda IPLB-SF-21 cells by adsorptive endocytosis. AcV1, a neutralizing monoclonal antibody, reacts with the 64K protein and in doing so prevents efficient entry. In this communication we report that treatment of AcMNPV BV with either trypsin or proteinase K cleaves the 64K protein into one major fragment of 34.6K and two minor fragments of 36K to 37.2K that are retained with the virus. All of the fragments are glycosylated. Protease treatment does not reduce viral infectivity, but it does result in the destruction of the AcV1-reactive epitope; thus AcV1 is not able to neutralize protease-treated AcMNPV BV. Polyclonal antiserum to BV is able to recognize both cleaved and uncleaved 64K and neutralize both protease-treated and untreated virus. Protease treatment does not diminish the sensitivity of AcMNPV BV to chloroquine, but it does cause the virus to become more susceptible to inactivation by 2-mercaptoethanol (2-ME) even though exposure to 2-ME does not result in dissociation of the fragments from the virus.

Chloroquine↗
PubMed1980

Titration of a cytoplasmic polyhedrosis virus by a tissue microculture assay: some applications.

A simple tissue microculture technique was developed for the titration of a cytoplasmic polyhedrosis virus (CPV) from Euxoa scandens. The procedure was similar to the 50% tissue culture infectious dose assay, but a single infected cell, detected by the presence of cytoplasmic polyhedra, was scored rather than the degeneration of cell monolayers. The filtration of CPV suspensions resulted in decreased virus titers under certain conditions. This microculture assay was used to determine the effect of cell disruption methods on virus yields. Sonication of infected cells was more efficient than freeze-thawing for the recovery of nonoccluded virus.

Animals↗
PubMed1985

Boolarra virus: a member of the Nodaviridae isolated from Oncopera intricoides (Lepidoptera: Hepialidae).

A small RNA virus with a bipartite genome was isolated from Oncopera intricoides. It was named Boolarra virus (BoV). The particle had a diameter of 30 nm, a sedimentation coefficient of 140S, and a buoyant density of 1.34 g/ml in CsCl. The capsid proteins were examined by SDS-PAGE and consisted of a major species of molecular weight (mol. wt.) 38,000 and a minor one of mol. wt. 40,600. The particle contained 21.2% RNA which was divided between a 22S and a 17S species whose mol. wts. were 1.03 x 10(6) and 0.47 x 10(6), respectively. These properties were comparable to those of the Nodaviridae and established BoV as a member of the family. Serologically, BoV shared antigenic determinants with both Nodamura virus and black beetle virus.

Animals↗