Abnormal Formation of Polyhedra Resulting from a Single Mutation in the Polyhedrin Gene of Autographa californica Multicapsid Nucleopolyhedrovirus
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Polyhedrin
Autographa californica
Sf9
Wild type
Recombinant virus
The baculovirus expression vector system (BEVS), utilizing the Autographa californica nuclear polyhedrosis virus (AcNPV), has turned out to be an attractive alternative for high-level expression (<600 mg/l) of recombinant proteins. However, there is a shortage of reliable methods for monitoring the infection process in situations where marker proteins cannot be used.Three recombinant baculoviruses, FastBac1-wtGFP, VTBac-GFP, and VL1392-hIL-2Ralpha, all having the genes inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV), were used to infect Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-MB-0503) insect cells. The infection process of the recombinant baculoviruses was monitored by flow cytometric side-scatter and fluorescence intensity analyses over a period of 6-96 h.A clear correlation between the side-scatter (SSC) signal and the relative fluorescence was observed for both of the infected cell lines, compared to noninfected cells. Comparison of SSC histograms from noninfected insect cells with cells infected with the nonfluorescent recombinant baculovirus VL1392-hIL-2Ralpha showed a clear increase of SSC for the infected cells.The SSC parameter can therefore be utilized for flow cytometric monitoring of a baculovirus infection process in situations where suitable markers are not available.
Polyhedrin
Autographa californica
Sf9
Nuclear Polyhedrosis Virus
Baculoviridae
Recombinant virus
mCherry
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Exigua
Autographa californica
Sf9
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Increasing evidence sugggest that in addition of balculovirus controling insect host, host cells also responds to balculovirus infection. However, compared to existing knowledge on virus gene, host cell responses are relatively poorly understood.In this study, Spodoptera frugiperda (Sf9) cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The protein composition and protein changes of Spodoptera frugiperda (Sf9) cells of different infection stages were analysed by isobaric tag for relative and absolute quantification (iTRAQ) techniques.A total of 4004 Sf9 proteins were identified by iTRAQ and 413 proteins were found as more than 1.5-fold changes in abundance. The 413 proteins were categorised according to GO classification for insects and were categorised into: biological process, molecular function and cellular component.The determination of the protein changes in infected Sf9 cells would help to better understanding of host cell responses and facilitate better design of this virus-host cell interaction in pest insect control and other related fields.
Sf9
Autographa californica
Baculoviridae
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Polyhedrin
Autographa californica
Nuclear Polyhedrosis Virus
Hemolymph
Recombinant virus
Baculoviridae
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A novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (ACNPV) producing the green fluorescent polyhedra was constructed and characterized. The recombinant virus was stably produced fluorescent polyhedra in the infected cells and the morphology of the polyhedra was nearly similar to that of wild-type AcNPV. For the production of the fluorescent polyhedral the green fluorescent protein (GFP) gene was introduced under the control of polyhedrin gene promoter of AcNPV by translational fusion in the front and back of intact polyhedrin gene. The recombinant baculovirus was named as CXEP, As expected, the 93 kDa fusion protein was expressed in the CXEP-infected cells. Interestingly, however, the cells infected with CXEP also showed a 33 kDa protein band as cells infected with wild-type AcNPV. The results of Southern blot analysis and plaque assay suggested that two types of baculoviruses expressing the GFP fusion protein or only native polyhedrin were formed through homologous recombination between two polyhedrin genes in the same orientation. Thus, this system can be applied for the production of recombinant polyhedra with foreign gene product of diverse interest.
Polyhedrin
Autographa californica
Nuclear Polyhedrosis Virus
Recombinant virus
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We have constructed a recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), containing green fluorescent protein (GFP) gene from the jellyfish, Aequorea victoria, and transferred it into the domestic silkworm Bombyx mori larvae for the production of visible transgenic silkworm of living organism. When one day-old fifth instar female larvae were injected with the recombinant AcNPV of 1x10 plaque forming units, the bright glow of GFP was detected in the recombinant AcNPV-infected larvae and in the newly hatched larvae of the next generation. Our findings demonstrate that the viral replication was detected in the silkworm treated with the recombinant ACNPV and the gfp gene was expressed under the transcriptional control of the polyhedrin gene promoter, Furthermore, the gfp gene was transmitted to the next generation, suggesting that this system can be applied for the development of transgenic silkworms.
Autographa californica
Polyhedrin
Nuclear Polyhedrosis Virus
Recombinant virus
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The <i>Autographa californica</i> multinucleocapsid nuclear polyhedrosis virus (AcMNPV) has a broad host range among Lepidoptera. In contrast, the <i>Spodoptera exigua</i> MNPV (SeMNPV) can replicate efficiently only in <i>S. exigua</i> larvae or <i>S. exigua</i>-derived cell lines. In this study, we examined the coinfection of <i>S. exigua</i> Se301 and <i>Spodoptera frugiperda</i> IPLB-SF21AEII (Sf21) cell lines with SeMNPV and AcMNPV recombinant (Ac360-<i>501β-gal</i>) which was constructed for expression of β-galactosidase under control of the polyhedrin promoter. Coinfection led to the restriction as the level of late gene expression, nonoccluded virus production, and DNA replication of Ac360-<i>501β-gal</i> in both Se301 and Sf21 cell lines. In contrast, Ac360-<i>501β-gal</i> supported the SeMNPV replication in Sf21 cells. Occurrence of recombinants, between Ac360-<i>501β-gal</i> and SeMNPV, with expanded host range was not observed in coinfected Sf21 cells. This suggests that Ac360-<i>501β-gal</i> supports the SeMNPV replication through <i>trans</i>-activation.
Autographa californica
Polyhedrin
Exigua
Nuclear Polyhedrosis Virus
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We described a baculovirus expression system for high level production of secreted murine recombinant IL-4. We have constructed a recombinant baculovirus based on Autographa californica polyhedrosis virus, containing both a synthetic PCR-derived murine IL-4 cDNA under the control of the polyhedrin promoter and the lacZ gene under the control of the P10 promoter to allow an easy detection of recombinant virus. The baculovirus IL-4 was fully functional in biological assay and was present under two glycosylated forms in the supernatants of infected Sf9 cells. We also detected a third unglycosylated intracytoplasmic form resulting from a fusion between the 35 first amino acids of polyhedrin and the murine IL-4. Finally, confocal microscopy showed that this recombinant protein was secreted along a classical pathway like in mammalian cells.
Polyhedrin
Autographa californica
Sf9
Baculoviridae
Nuclear Polyhedrosis Virus
Recombinant virus
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Polyhedrin
Autographa californica
Sf9
Exigua
Polyhedron
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Citations (3)
The polyhedrin gene(polh) of Autographa californica multicapsid nuclear polyhedrosis virus(AcMNPV) was cloned into the recombinant BmNPV(API4) genome which the polyhedrin gene of Bombyx mori nuclear polyhedrosis virus(BmNPV) was replaced by API4. And a recombinant BmNPV(OAc API4) was obtained. The recombinant virus was able to produce AcMNPV polyhedrin protein and expressed the Arrowhead proteinase inhibitor. The budded virus of the recombinant BmNPV(OAc API4) was able to infect BmN cells, and silkworm larvae using injection while per os inoculation with occluded virus couldn't infect the larvae. It was indicated that per os inoculation of NPV might be concerned with the type of polyhedrin proteins.
Polyhedrin
Nuclear Polyhedrosis Virus
Autographa californica
Recombinant virus
Infectivity
Baculoviridae
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