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At least 865 records · Page 48Linked to original sources
PubMed1990

Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was injected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein. The recombinant human adenosine deaminase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins.

Adenosine Deaminase↗
PubMed1999

1995 epizootic of vesicular stomatitis (New Jersey serotype) in the western United States: an entomologic perspective.

Entomologic and epizootic data are reviewed concerning the potential for transmission of vesicular stomatitis (VS) virus by insects, including field data from case-positive premises in New Mexico and Colorado during the 1995 outbreak of the New Jersey serotype (VSNJ). As with previous outbreaks of VSNJ in the western United States, the 1995 epizootic illustrated that risk of exposure is seasonal, increasing during warm weather and decreasing with onset of cool weather; virus activity spread from south to north along river valleys of the southwestern and Rocky Mountain states; clinical disease was detected most commonly in horses, but also occurred in cattle and 1 llama; and most infections were subclinical. Overall, 367 case-positive premises were identified during the 1995 outbreak, with foci of virus activity along the Rio Grande River south of Albuquerque, NM, in southwestern Colorado, and along the Colorado River near Grand Junction, CO. The establishment of a 16-km (10-mile) radius zone of restricted animal movement around confirmed positive premises, along with imposition of state and international embargoes, created economic hardship for livestock owners and producers. The importance of defining the role of blood-feeding insects as biological vectors of VSNJ virus relative to risk factors that promote high levels of insect transmission, such as the presence of livestock along western river valleys, blood feeding activity, and frequent transport of animals for recreational purposes, is emphasized as a basis for developing effective disease management.

Animals↗
PubMed1992

Influenza A virus NP protein expressed in insect cells by a recombinant baculovirus is associated with a protein kinase activity and possesses single-stranded RNA binding activity.

Influenza A virus NP protein, the phosphoprotein associated with viral RNA in ribonucleoprotein (RNP) complexes, has been expressed at high levels (approximately 100 mg/liter cells) in insect (Sf9) cells by a baculovirus recombinant, and was localized almost entirely in the nuclei of these cells. NP was purified by immuno-affinity chromatography, and purified NP was shown to autophosphorylate and to phosphorylate casein in a cAMP-independent reaction. Furthermore, purified NP was able to bind to ssRNA as demonstrated by a mobility shift of ssRNA in non-denaturing gels. The binding of NP to ssRNA caused a diminution of its kinase activity in proportion to binding.

Animals↗
PubMed1981

Plaque assay of Heliothis zea baculovirus employing a mixed agarose overlay.

The nuclear polyhedrosis virus of Heliothis zea has been titrated in Heliothis zea cells by the plaque method, using 1 percent mixed agarose containing a mixture of Seakem and Ultra pure agarose. Visible plaques, formed 8 days postinfection, ranged in diameter from 0.5 to 2 mm. Dose-response experiments indicated that a single particle initiated the formation of a plaque. The titration of Heliothis zea baculovirus by the newly described plaque method provides an accurate technique for the determination of virus concentration.

Animals↗
PubMed1985

DNA homology relationships between Spodoptera littoralis nuclear polyhedrosis virus and other baculoviruses.

The DNA sequence homology relationships between Spodoptera littoralis nuclear polyhedrosis virus (NPV) and five other lepidopteran NPVs were studied by hybridization of 32P-labeled NPV DNAs to Southern blots of restriction-endonuclease-digested NPV DNA. The S. littoralis NPV (SlMNPV) shows extensive DNA sequence homology through most of the genome with S. frugiperda NPV and S. exigua NPV. Heliothis armigera NPV, H. zea NPV, and Autographa californica NPV (AcMNPV) share fewer regions of homology with SlMNPV. Using the cloned HindIII fragment V of AcMNPV DNA, fragments with sequence homology to the polyhedrin gene of AcMNPV were identified for the viruses studied.

DNA Restriction Enzymes↗
PubMed2001

A comparison of techniques for detecting Invertebrate iridescent virus 6.

The aim of this study was to compare the sensitivity and precision of various methods for the detection and quantification of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae) isolated from a the stem-boring moth Chilo suppressalis, and to apply these techniques to the detection of covert infections in the wax moth, Galleria mellonella. The relationship between the virus concentration and absorbance at 260 nm was linear over the range of 1.6 x 10(9)-5.6 x 10(10) particles/ml. TCID(50) assays using 12 different cell lines indicated that two Drosophila lines, DL2 and DR1, had the highest susceptibility whereas cell lines from Aedes albopictus and Plutella xylostella were four orders of magnitude less sensitive. TCID(50) values for IIV-6 in Spodoptera frugiperda Sf9 cells gave the particle-infectivity ratios of 15-64 virus particles/IU. An insect bioassay involved injecting doses of 1-100 IIV-6 particles into the third instar G. mellonella larvae. The prevalence of patent infection was 20-26% at a dose of 1 particle per larva rising to 86-92% at 10 particles and 100% at doses of 50 or 100 particles. Of the insects that survived to adulthood, between 5.8 and 75% caused patent infections when injected into G. mellonella larvae, indicating that they were covertly infected. A PCR technique resulted in 95% detection at 1000 virus particles per insect. Of the insects that proved positive for covert infection by insect bioassay, 41% also proved positive by PCR analysis. It is concluded that the G. mellonella bioassay is highly reliable for detection of doses of 10 particles or more and for determining the relative activity of IIV-6 preparations at doses as low as 1 particle per insect. PCR had a slightly lower sensitivity followed by the insect cell culture assay.

Aedes↗
PubMed1992

Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence.

Recently, a parvo-like virus was newly isolated from silkworm larvae and the two viral DNAs (VD1 and VD2) with different electro-mobilities were identified. We cloned the viral DNAs in a plasmid pUC119 and demonstrated that these two DNAs were not a bimorphic molecules though they shared a common terminal sequence of 53 nucleotides. In addition, the sequence at the 5' terminus of each strand of the viral DNA was located in inverted form at its 3' terminus. On the other hand, the nucleotide sequences of VD1 and VD2 were different from that of the Bombyx densovirus (Ina isolate) DNA.

Animals↗
PubMed1992

Characterization of functional human erythrocyte-type glucose transporter (GLUT1) expressed in insect cells using a recombinant baculovirus.

The human erythrocyte-type glucose transporter (GLUT1) has been abundantly expressed in insect cells by using a recombinant baculovirus. At 4 days after infection with the virus, the insect cell-surface and intracellular membranes were found to contain greater than 200 pmol of D-glucose-sensitive binding sites for the transport inhibitor cytochalasin B per mg of protein. The characteristics of binding were identical with those of the erythrocyte transporter, although the two proteins differed substantially in apparent Mr, probably as a result of glycosylation differences.

Animals↗
PubMed1976

An iridovirus from bees.

An iridovirus, Apis iridescent virus (AIV), isolated from sick adult specimens of Apis cerana (Hymenoptera) from Kashmir, closely resembles iridescent viruses from Tipula and Sericesthis spp. (TIV and SIV). However, AIV is only distantly related serologically to TIV and SIV and is even more remotely related to several other similar viruses that were tested in tube precipitation tests with intact particles. AIV multiplies in Apis mellifera, forming cytoplasmic iridescent crystalline aggregates in several tissues, but unlike all the other iridoviruses tested, it failed to multiply in Galleria mellonella.

Animals↗
PubMed1989

Nucleotide sequence of the polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus A strain with nuclear localization of polyhedra.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) strain H produces many hexahedral polyhedra (inclusion bodies) in the cytoplasm of insect midgut epithelial cells. The mutant A strain, however, produces polyhedra in the nucleus. We determined cDNA sequences of the polyhedrin genes, the smallest of the 10 genome segments, of these two strains. The polyhedrin genes of the H and A strains were 944 bp long, and encoded polypeptides of 248 amino acids (Mr 28,500) and 252 amino acids (Mr 29,000), respectively. The extra four amino acid residues at the carboxy terminus of the strain A polyhedrin (Arg-Leu-Leu-Val) were the result of a single nucleotide substitution at an opal stop codon (TGA----CGA). A further amino acid substitution of the histidine residue at position 101 (His----Tyr) was seen. The carboxy-terminal extension revealed a considerable similarity to the consensus amino acid sequence of the DNA-binding domain of many DNA-binding proteins. We discuss the relationship between the intracellular localization of polyhedrins and mutations in their amino acid sequences.

Amino Acid Sequence↗
PubMed2004

Analysis of the replication kinetics of the ChimeriVax-DEN 1, 2, 3, 4 tetravalent virus mixture in Aedes aegypti by real-time reverse transcriptase-polymerase chain reaction.

The vector competence of mosquitoes for chimeric viruses being developed as vaccines to protect against dengue (DEN) virus infection were evaluated in a cooperative agreement with Acambis, Inc. Chimeric viruses have been constructed that contain the premembrane (prM) and envelope (E) genes of each of the wild-type (wt) DEN virus serotypes, DEN-1, DEN-2, DEN-3, and DEN-4, in the yellow fever (YF) vaccine virus (strain 17D) YF-VAX backbone. It was previously shown that the replication profile of ChimeriVax-DEN2 virus in Aedes albopictus C6/36 cells and in vivo in Ae. aegypti mosquitoes corresponded to that of YF-VAX virus; replication was restricted in C6/36 cells, and Ae. aegypti were poorly infected via an artificial infectious blood meal. Thus, there is very little risk of transmission by mosquitoes of ChimeriVax-DEN2 vaccine virus through the bite of a mosquito. However, because ChimeriVax-DEN 1, 2, 3, 4 viruses will be administered to humans simultaneously, growth of a mixture of ChimeriVax-DEN 1, 2, 3, 4 viruses was assessed in both C6/36 cells in culture and in the Ae. aegypti mosquito, which is the primary vector of both YF and DEN viruses. Mosquitoes were intrathoracically (IT) inoculated with virus or fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN 1, 2, 3, 4 were compared with the wt DEN and YF-VAX viruses. A quantitative real-time reverse transcriptase-polymerase chain reaction assay was developed as a method to detect and differentiate replication of each of the four ChimeriVax-DEN serotypes in the ChimeriVax-DEN 1, 2, 3, 4 tetravalent mixture. Growth of the chimeric viruses in C6/36 cells and in IT-inoculated Ae. aegypti was lower than that of YF-VAX virus; in previous studies Ae. aegypti was shown to be refractory to infection by YF-VAX virus. The growth rate of each chimeric virus was similar whether it was a single serotype infection, or part of the tetravalent mixture, and no interference by one chimeric virus over another chimeric serotype was observed. ChimeriVax-DEN viruses infected mosquitoes poorly via an infectious blood meal compared with wt DEN viruses. Therefore, it is unlikely that a mosquito feeding on a viremic vaccinee, would become infected with the chimeric viruses. Thus, there is very little potential for transmission by mosquitoes of the ChimeriVax-DEN vaccine viruses.

Aedes↗
PubMed1988

Purification and functional properties of simian virus 40 large and small T antigens overproduced in insect cells.

The insect baculovirus Autographa californica nuclear polyhedrosis virus was used as an expression vector for the simian virus 40 (SV40) small t (t) and large T (T) antigens. Spodoptera frugiperda (SF9) cells infected with recombinant viruses encoding these proteins produced approximately 1 to 2 micrograms of t and up to 30 micrograms of T per 3 X 10(6) cells. The former was highly soluble after Nonidet P-40 extraction of the infected cells, unlike its Escherichia coli-produced counterpart. Both SF9-produced proteins were of authentic size and could be readily immunoprecipitated by specific antibodies. Single-step immunoaffinity chromatography was used to purify the two proteins to near homogeneity, with yields averaging 70% in each case. Experiments to test the biological activity of the baculovirus SV40 proteins showed that SF9 t was capable of associating with two of the cellular proteins reported to bind to t in SV40-infected mammalian cells. Moreover, SF9 T had ATPase activity comparable to that of T produced in monkey cells, exhibited helicase activity and SV40 origin-specific DNA binding, and was active in the SV40 DNA replication assay in vitro. Thus, the SV40 T antigens produced in insect cells can be used in future studies of their biochemical roles in vitro and in vivo.

Adenosine Triphosphatases↗