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At least 685 records · Page 38Linked to original sources
PubMed1999

Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate.

BACKGROUND & AIMS: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. Successful vaccine development is crucial in controlling global HCV infection. We have previously described the generation of HCV-like particles (HCV-LPs) in insect cells using a recombinant baculovirus containing the complementary DNA of the HCV structural proteins. These HCV-LPs had similar morphological and biophysical properties as the putative virions. In this study, we analyzed the structural features, antigenic composition, seroreactivity, and immunogenicity of purified HCV-LPs. METHODS: HCV-LPs were analyzed by electron microscopy and antibody immunolabeling and precipitation. An enzyme-linked immunosorbent assay (ELISA) using HCV-LPs was developed. The humoral response to HCV-LPs in mice was studies by core and envelope ELISAs, Western immunoblotting, and immunofluorescence. RESULTS: Structural and antigenic compositions of HCV-LPs were shown to be similar to those of putative HCV virions. Using the HCV-LP ELISA, high-titer anti-HCV antibodies were detected in individuals infected with various HCV genotypes. In vivo, HCV-LPs elicited a humoral response broadly directed against HCV structural proteins. CONCLUSIONS: HCV-LPs resemble HCV virions and are capable of inducing a humoral response targeted against various regions of HCV structural proteins, suggesting that HCV-LPs may be promising as a potential vaccine candidate.

Animals↗
PubMed1980

Viral pesticides : biohazard evaluation on the cytogenetic level.

Promising agents for integrated pest control are naturally occuring insect pathogenic viruses that belong to the group of baculoviruses. We have performed experiments using mammalian cells from different species in vivo and in vitro to test a possible effect of baculoviruses on chromosome aberration rates and sister chromatid exchanges. No cytogenetic effects were found.

Animals↗
PubMed1998

Immune response to recombinant visna virus Gag and Env precursor proteins synthesized in insect cells.

Two different recombinant visna virus (VV) gag-baculoviruses were constructed for the expression of precursor VV Gag in insect cells. Both recombinant Gag viruses expressed proteins migrating on SDS PAGE at the predicted rate for VV Gag precursor, Pr50gag. However, differences were seen in the morphology of the virus-like particles produced. Monoclonal antibody directed against the VV Gag capsid protein (p25) and sera from sheep infected with ovine lentiviruses reacted to both 50-kDa proteins. A recombinant VV env-baculovirus was constructed, substituting sequences encoding the signal peptide of VV Env with the murine IFN-gamma analogue. Sera from ovine lentivirus infected sheep reacted in immunoblots with two proteins of approximately 100 and 200 kDa found in the plasma membrane of insect cells infected with env-recombinant virus. Sheep immunized with either the recombinant Gag or the Env proteins developed high antibody titers to VV in ELISA. The serum of sheep and ascitic fluid of mice immunized with the recombinant Gag reacted with native Pr50gag and the processed Gag proteins in immunoblots, whereas serum of the recombinant Env immunized sheep reacted with VV gp135 and a putative oligomer of gp135. The immunized sheep responded specifically to visna virus by lymphocyte proliferation in vitro.

Animals↗
PubMed1994

Phylogenetic characterisation of bluetongue viruses from naturally-infected insects, cattle and sheep in Australia.

The polymerase chain reaction was used to detect the presence of bluetongue virus (BTV) in a number of clinical and insect samples collected in the Northern Territory of Australia. Sequence analyses of the amplified BTV genes differentiated endemic Australian and exotic viruses. Two potential exotic BTV were detected as a result of PCR analyses of blood from sentinel animals and of the insect vector, Culicoides wadai. The detection of BTV in C wadai was the first direct demonstration of the presence of BTV in this potential vector. This new technology can significantly reduce the time taken for a diagnosis from a clinical sample and increase the amount of useful information obtained on a BTV isolate by using rapid sequencing techniques. Sequence data were used to differentiate between BTV20 isolated in 1975 and two isolates of the same serotype, isolated in 1992, and indicated that the latter were probably a recent incursion into Australia from Indonesia due to their greater VP3 sequence homology to the BTV9 (Java) than to Australian BTV isolates.

Animals↗
PubMed1982

The use of a protein A conjugate in an indirect enzyme-linked immunosorbent assay (ELISA) of four closely related baculoviruses from Spodoptera species.

The virus particles from four closely related insect nuclear polyhedrosis virus (NPVs) isolated from their homologous Spodoptera spp. hosts were examined using an indirect enzyme-linked immunosorbent assay (ELISA), employing both swine anti-rabbit and protein A conjugate. Results from both systems indicated that S. littoralis NPV is serologically distinct from the other three viruses, S. frugiperda NPV, S. exempta NPV and S. exigua NPV, which are closely related. The relatedness of these viruses is discussed with reference to existing knowledge and the results obtained from both conjugate systems.

Antigens, Viral↗
PubMed1991

Expression of hepatitis B virus surface antigen gene in insect baculovirus vector systems.

The gene coding for the hepatitis B virus surface antigen (HBsAg) under the control of Autographa californicanuclear polyhedrosis virus polyhedrin promoter was successfully inserted into the genome of the Trichoplusia ni nuclear polyhdrosis virus. Infection of Spodoptera frugiperda cells with this recombinant virus produced a significant amount of HBsAg protein and secreted 22 nm particles containing the HBsAg. The expression of HBsAg gene was also obtained both in Trichoplusia ni larvae and in Philosamia cynthia ricini prepupae when infected with the recombinant virus. The HBsAg proteins expressed by baculovirus vector systems have morphological and antigenic properties identical to the 22 nm particles secreted by human cells.

Animals↗
PubMed2004

The role of food plant and pathogen-induced behaviour in the persistence of a nucleopolyhedrovirus.

Insect baculoviruses can survive between epidemics as infectious particles external to the host. Many pathogens persist in reservoirs, i.e., microhabitats where survival is enhanced, for example due to protection from the degrading effects of UV irradiation. However, the probability of infecting new susceptible hosts is usually reduced. Persistence of pathogens and their movement in and out of reservoirs is an important, albeit little understood, aspect of insect pathogen ecology. This study investigated interactions between the behaviour of infected insect hosts, virus distribution and plant species on the persistence of the winter moth (Operophtera brumata) nucleopolyhedrovirus. Habitat influenced the persistence of infectious baculovirus in the field: virus on Sitka spruce (Picea sitchensis) and oak (Quercus robur) in forested areas retained more infectivity than virus on heather (Calluna vulgaris) in an unshaded habitat. Plant species per se did not directly affect the persistence of virus on the foliage of potted seedlings. Virally infected insects had altered behaviour and moved down plants relative to control insects, whereas in other systems larvae show height-seeking behaviour. Consequently, the majority of virus particles were distributed on plant stems. In two experiments (one using winter moth NPV and one Mamestra brassicae NPV) virus persisted better on plant stems relative to foliage. Neonate larvae were shown to be able to acquire infections from tree stems contaminated with a low level of virus. These data suggest that plant stems may be important reservoirs for between-year persistence of this pathogen. The observed virus-induced changes in host behaviour in winter moth could enhance the viral persistence by increasing the deposition of occlusion bodies in these reservoirs.

Animals↗
PubMed1980

Mass production of insect cells in suspension.

The production of insect pathogenic viruses for the use in integrated pest control is presently done in vivo in populations of their natural hosts. However, efforts are made in several laboratories to produce some of these baculoviruses in insect cell cultures. The mass production of the viruses in vitro depends on a technology for the mass production of cells. Data are presented for the production of up to 10(10) insect cells per logarithmic growth phase in a continuously running fermenter culture at volumes of 9-10 litres.

Animals↗
PubMed1997

Characteristics of acyrthosiphon pisum virus, a newly identified virus infecting the pea aphid.

A new virus was isolated from the pea aphid, Acyrthosiphon pisum, and tentatively named Acyrthosiphon pisum virus (APV). The isometric virus particles were approximately 31 nm in diameter and contained a single-stranded RNA molecule of approximately 10 kb. Four structural proteins were observed with molecular masses of approximately 23.3, 24.2, 34.5, and 66.2 kDa. The 34.5-kDa capsid protein was the most abundant product in purified virions. Computer-assisted analysis revealed no significant homology between an internal sequence of 37 amino acids of the 34.5-kDa protein of APV and other polypeptides of viral origin. APV was not immunologically related to other ssRNA viruses from hemipteroid insects, such as aphid lethal paralysis virus, Rhopalosiphum padi virus, and Nezara viridula virus type 1. Immunolocalization on ultrathin sections of 3-day-old nymphs of A. pisum showed that APV antigen was predominantly present in the epithelial cells of the digestive tract. Virus particles were also observed associated with the microvilli of the intestine. Occasionally, muscle cells and mycetocyte cells were found infected. Purified APV, fed to 1-day-old A. pisum nymphs, significantly reduced the growth of the aphid and increased the time needed to reach maturity.

Amino Acid Sequence↗
PubMed2003

[Expression and purification of varicella-zoster virus glycoprotein I gene in insect cells].

OBJECTIVE: To express the cloned gene glycoprotein I (gpI) of varicella-zoster virus (VZV), Beijing VZV 84-7 strain in insect cells and to purify its expression product. METHODS: The gene coding for gpI of VZV was amplified from viral DNA by PCR and cloned into baculovirus transfer vector (pBacPAK9), and recombinant transfer vector plasmid pBacVZVgpI was obtained. The inserted gpI gene in the pBacVZVgpI was sequenced. Insect cells Sf 9 were co-transfected with the recombinant transfer vector plasmid pBacVZVgpI and wild type linear baculovirus BacPAK6 (digested with Bsu36I) DNA. The recombinant baculoviruses containing the VZV 84-7 gpI gene was isolated through several rounds of limited dilution. Recombinant protein gpI was expressed in insect cells Sf 9, postinfected with recombinant baculoviruses. The expressed recombinant gpI was purified by lectin affinity chromatography and its antigenicity and immunogenicity were investigated. RESULTS: The gene coding for gpI of VZV was obtained by PCR and the gpI gene of pBacPAK9 was confirmed by DNA sequencing. The recombinant gpI was expressed in insect cells Sf 9, post-infected with recombinant baculovirus and identified by SDS-PAGE and western blotting, with its product in cell culture reaching the peak in 72 hours and with a molecular mass of 58 kd and 70 kd, the same as theoretical values. Results of immunoassay with cell lysates infected by recombinant baculoviruses indicated that recombinant protein expressed in insect cells had ability of eliciting specific antibodies against native VZV in mice and complement-dependent neutralizing antibodies. The purified recombinant gpI gave a product with a purity of more than 80%. ELISA and Western-blot analysis demonstrated that purified protein had specific VZV antibody-binding activity. This suggested that the recombinant gpI expressed in insect cells had the same biological characteristics as its native counterpart. CONCLUSION: Baculovirus-insect cells could be used to express the gene of VZV gpI, which could provide a basis for quantitative analysis of VZV antigen, and preparation of its subunit vaccine.

Animals↗
PubMed2006

Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies.

Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.

Animals↗
PubMed1994

Quantification of latent Mamestra brassicae nuclear polyhedrosis virus in M. brassicae insects using a PCR-scintillation proximity assay.

A laboratory culture of Mamestra brassicae insects (MbLC) was found to harbour a latent baculovirus infection. The copy number of the occult MbNPV genome in both the MbLC larvae, and in a cell line derived from the fat body of MbLC was determined by the use of a rapid and convenient PCR-scintillation proximity assay (SPA). The SPA system relies on the use of fluomicrospheres (SPA beads) coated with acceptor molecules which are capable of binding radiolabelled ligands in solution. In the assay described, a biotinylated PCR primer is used and [3H]dNTPs are incorporated into the amplified DNA. The SPA beads are coated with streptavidin, and after binding the biotinylated primer, any amplified, radiolabelled DNA will activate the fluor. The amount of amplified DNA from the target sequence can then be directly quantified using a scintillation counter. The number of MbNPV genomes present in a persistently infected M. brassicae cell, as proposed by SPA, suggest between 13 and 20 copies of the viral genome may be present in individual fat body cells.

Animals↗
PubMed1998

Expression of bovine leukemia virus ENV glycoprotein in insect cells by recombinant baculovirus.

The gp51-p30 glycoprotein constituting BLV envelope was expressed in Sf-21 insect cells by means of recombinant baculoviruses. Post-infection cell lysates were analyzed, in order to define the immunologic reactivity of recombinant products. Oligosaccharide chains, containing N-acetylglucosamine, mannose, galactose and sialic acid were found on recombinant gp51-p30. In order to investigate the timing of transcription and translation of the glycoprotein, kinetic assays were carried out on cell lysates and directly in situ on Sf-21 cells during the course of baculovirus infection. The use of different solubilizing reagents was also evaluated in order to rescue recombinant glycoprotein from its subcellular location.

Animals↗