scholarly journals Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin

2021 ◽  
Vol 104 (1) ◽  
pp. 003685042110042
Author(s):  
Swarnendu Basak ◽  
Hae-Ji Kang ◽  
Ki-Back Chu ◽  
Judy Oh ◽  
Fu-Shi Quan
Keyword(s):  
Post Infection ◽  
Plaque Assay ◽  
Recombinant Baculovirus ◽  
Accurate Method ◽  
Surface Proteins ◽  
Recombinant Baculoviruses ◽  
Sf9 Cells ◽  
Influenza Hemagglutinin ◽  
Infected Cells ◽  
Plaque Quantification

Recombinant baculoviruses (rBVs) have been extensively used to generate virus-like particles, and baculoviruses expressing antigenic proteins have become efficient tools for inducing protective immunity. However, current methods for generating baculoviruses are costly and inefficient. Thus, the development of a simple, rapid, and accurate method of baculovirus titration is critically important. We established a method of plaque assay using an immunostaining method by which plaques can be easily visualized in Sf9 cells under a light microscope. Sf9 cells were infected with recombinant baculoviruses expressing influenza hemagglutinin surface proteins from H1N1 (A/California/04/09) or rH5N1 (A/Vietnam/1203/04). The infected cells were incubated with anti-HA antibody and the plaques were visualized using the chromogen 3′3-diaminobenzidine (DAB). Plaques were observed from days 1 to 6 post-infection, and differences in Sf9 cell seeding densities resulted in variations in the final plaque quantification. Sf9 cells seeded at a concentration of 5.5 × 104 cells/well or 7.5 × 104 cells/well showed the higher plaque titers at days 3, 4, and 5 post-infection than those found at days 1, 2, and 6 post-infection. With 5.5 × 104 cells/well or 7.5 × 104 cells/well of cell concentrations, recombinant baculovirus for rBV-HA (H1N1) showed 6 × 107 pfu/ml of titer and rBVs for rBV-HA (rH5N1) showed 5.4 × 107 pfu/ml of titer. Three days of baculovirus incubation with a certain concentration of Sf9 cells seeded are required for a rapid, simple, and accurate plaque assay, which could significantly contribute to all baculovirus-related studies.

scholarly journals Volume and surface changes in vero cell and its nucleus after infection with measles virus: a stereological study

2011 ◽  
Vol 2 (1) ◽  
pp. 18
Author(s):  
Ali Noorafshan ◽  
Mohammad Motamedifar ◽  
Saied Karbalay-Doust
Keyword(s):  
Vero Cell ◽  
Post Infection ◽  
Measles Virus ◽  
Plaque Assay ◽  
Vero Cells ◽  
Uninfected Control ◽  
Mean Cell Volume ◽  
Significant Difference ◽  
Infected Cells ◽  
The Mean

Measles virus has no or indistinctive cytopathic effects (CPE) in cell couture system. Employment of some detecting methods like plaque assay or stereologic experiments, as a method of detecting of viral infection in the cells would be applicable. The aim of this study was investigating the early changes in quantitative parameters of measles virus infected Vero cells. Stereological methods using invariator, were applied for the first time to estimate cell and nucleus volume and cell surface of the infected Vero cell line with the measles virus.This method can be applied on other cultured cells.Vero cells grown in tissue culture plates for 48 hours at 36˚C were infected with 100TCID50 of AiK strain of measles virus. Volume and surface of the infected Vero cells were studied at 4, 9 and 25 hours post infection along with uninfected control cells. The mean cell volume and surface of the cells infected with measles virus, increased ~87% and ~50%, respectively, 4 hours post-infection, as compared with the uninfected control. The nuclei did not show any differences. The mean parameters of infected cells in other time intervals showed no significant difference comparing with the control cells. Although there are other specific methods, stereology may be used as an integrated protocol to detect cytophatic changes of the measles virus infected cells early in the permissive cell culture system.

Phosphorylated baculovirus p10 is a heat-stable microtubule-associated protein associated with process formation in Sf9 cells

1992 ◽  
Vol 102 (4) ◽  
pp. 739-752
Author(s):  
S. Cheley ◽  
K.S. Kosik ◽  
P. Paskevich ◽  
S. Bakalis ◽  
H. Bayley
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Recombinant Baculovirus ◽  
Catalytic Subunit ◽  
Sf9 Cells ◽  
Wild Type ◽  
Heat Stable ◽  
Process Formation ◽  
Infected Cells ◽  
Kinase A

Insect ovarian Sf9 cells extend processes with complex morphologies when infected with a recombinant baculovirus encoding the catalytic subunit of protein kinase A. Within the shafts of the processes are abundant microtubules, which, in contrast to those in Sf9 cells expressing the microtubule-associated protein tau, are generally not organized into parallel bundles. During infection the late viral polypeptide p10 becomes phosphorylated by the protein kinase A catalytic subunit at its penultimate residue, Ser92. The expression or phosphorylation of other major host cell or viral polypeptides does not change, compared with polypeptides from a wild-type viral infection. Once phosphorylated, p10 associates with microtubules in the infected cells and may thereby play a role in process formation.

scholarly journals High-efficiency expression and characterization of the synaptic-vesicle monoamine transporter from baculovirus-infected insect cells

1998 ◽  
Vol 330 (2) ◽  
pp. 959-966 ◽  
Author(s):  
K. Michael SIEVERT ◽  
S. David THIRIOT ◽  
H. Robert EDWARDS ◽  
E. Arnold RUOHO
Keyword(s):  
Synaptic Vesicle ◽  
High Efficiency ◽  
Insect Cells ◽  
Expression System ◽  
Broad Band ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Sf9 Cells ◽  
Monoamine Transporter ◽  
Infected Cells

The full-length cDNA for the rat synaptic-vesicle monoamine transporter (VMAT2) containing a C-terminal polyhistidine epitope has been engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) insect cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed at levels of 7.8×106 transporters per cell, as assessed by [3H]dihydrotetrabenazine binding. A 1 l culture of infected cells produced approx. 15 nmol (900 μg) of transporter. rVMAT2 expressed in the Sf9 cells bound [3H]dihydrotetrabenazine with a KD of 31.2 nM and a Bmax of 19.9 pmol/mg. Two polypeptides of 55 and 63 kDa were identified using the photolabel, 7-azido-8-[125I]iodoketanserin ([125I]AZIK). Photoaffinity labelling of rVMAT2 by 1 nM [125I]AZIK was protectable by 10 μM tetrabenazine and 10 μM 7-aminoketanserin. Digitonin-solubilized VMAT2 was purified to greater than 95% homogeneity using immobilized Ni2+-affinity chromatography, followed by lectin (Concanavalin A) chromatography. The purified transporter migrates as a single broad band with a molecular mass of approx. 63 kDa, as analyzed by SDS/PAGE. The purified transporter retained the ability to bind ligands ([125I]AZIK and [3H]dihydrotetrabenazine). The purified VMAT2 bound [3H]dihydrotetrabenazine with a KD of 86.2 nM. As is the case with the monoamine transporter from bovine chromaffin granule membranes, purified VMAT2 is covalently modified by dicyclohexylcarbodi-imide (DCCD) and is specifically labelled by [14C]DCCD. This labelling is inhibited by tetrabenazine and ketanserin. These data indicate that VMAT2 can be overexpressed using the baculovirus expression system and purified.

Insect cells infected with a recombinant baculovirus express both O- and N-glycosylated forms of the rat glycoprotein hormone α-subunit

1996 ◽  
Vol 16 (2) ◽  
pp. 141-149 ◽  
Author(s):  
R Delahaye ◽  
P Berreur ◽  
R Salesse ◽  
R Counis
Keyword(s):  
Post Infection ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Chorionic Gonadotrophin ◽  
Biologically Active ◽  
Glycoprotein Hormones ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Infected Cells

ABSTRACT Glycoprotein hormones LH, FSH, TSH and chorionic gonadotrophin are heterodimers composed of two non-covalently associated subunits, a common α- and a specific β-subunit. A recombinant baculo-virus containing a cDNA encoding the α-subunit of rat glycoprotein hormones was constructed. Viral-infected cells expressed, 48 h post infection, 7–10mg immunoreactive α-glycopolypeptide/6×108 cells, of which 65·6% was able to associate with native LHβ and formed a biologically active heterodimeric hormone that bound to testicular receptors. The treatment with specific glycanases showed that the recombinant α-subunit was produced as two differently glycosylated forms; an Mr 23 000 form which contained exclusively N-linked carbohydrate units and another of Mr 25 000 which appeared to contain additional O-linked carbohydrate. Data demonstrated that the α-subunit was expressed by insect cells in a manner similar to that by mammalian pituitary gonadotropes producing both the N- and O-glycosylated forms although only the N-glycosylated α-subunit is known to be capable of associating with the β-subunit.

scholarly journals Homophilic binding of PTP mu, a receptor-type protein tyrosine phosphatase, can mediate cell-cell aggregation

1993 ◽  
Vol 122 (4) ◽  
pp. 961-972 ◽  
Author(s):  
SM Brady-Kalnay ◽  
AJ Flint ◽  
NK Tonks
Keyword(s):  
Fusion Protein ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Recombinant Baculovirus ◽  
Sf9 Cells ◽  
Homophilic Binding ◽  
Protein Tyrosine ◽  
Baculovirus Infection ◽  
Induced Aggregation ◽  
Cell Cell

The receptor-like protein tyrosine phosphatase, PTPmu, displays structural similarity to cell-cell adhesion molecules of the immunoglobulin superfamily. We have investigated the ability of human PTPmu to function in such a capacity. Expression of PTPmu, with or without the PTPase domains, by recombinant baculovirus infection of Sf9 cells induced their aggregation. However, neither a chimeric form of PTPmu, containing the extracellular and transmembrane segments of the EGF receptor and the intracellular segment of PTPmu, nor the intracellular segment of PTPmu expressed as a soluble protein induced aggregation. PTPmu mediates aggregation via a homophilic mechanism, as judged by lack of incorporation of uninfected Sf9 cells into aggregates of PTPmu-expressing cells. Homophilic binding has been demonstrated between PTPmu-coated fluorescent beads (Covaspheres) and endogenously expressed PTPmu on MvLu cells. Additionally the PTPmu-coated beads specifically bound to a bacterially expressed glutathione-S-transferase fusion protein containing the extracellular segment of PTPmu (GST/PTPmu) adsorbed to petri dishes. Covaspheres coated with the GST/PTPmu fusion protein aggregated in vitro and also bound to PTPmu expressed endogenously on MvLu cells. These results suggest that the ligand for this transmembrane PTPase is another PTPmu molecule on an adjacent cell. Thus homophilic binding interactions may be an important component of the function of PTPmu in vivo.

scholarly journals Characterization and purification of human retinoic acid receptor-γ 1 overexpressed in the baculovirus-insect cell system

1992 ◽  
Vol 287 (3) ◽  
pp. 833-840 ◽  
Author(s):  
A P Reddy ◽  
J Y Chen ◽  
T Zacharewski ◽  
H Gronemeyer ◽  
J J Voorhees ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Retinoic Acid Receptor ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Sf9 Cells ◽  
Acid Receptor ◽  
Gel Retardation

The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.

scholarly journals Ectopic Expression of Virus Innexins in Heliothis virescens Disrupts Hemocyte-Mediated Encapsulation and Host Viability

2019 ◽  
Author(s):  
Peng Zhang ◽  
Matthew W Turnbull
Keyword(s):  
Gene Family ◽  
Gap Junction ◽  
Host Range ◽  
Heliothis Virescens ◽  
Genome Structure ◽  
Ectopic Expression ◽  
Recombinant Baculovirus ◽  
Gene Families ◽  
Recombinant Baculoviruses ◽  
Susceptible Host

1.AbstractPolydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins, which are virus homologues of insect gap junction genes, the innexins. Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2, using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens. Following intrahemocoelic injections, we observed >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin-recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock infected larvae. Similarly, larvae infected with vinnexin-recombinant baculoviruses were less likely to molt relative to controls, and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. These results, in light of previous findings, support that Polydnavirus Vinnexin gene family members may provide complementary, rather than redundant, effects. This in turn indicates a need to test gene family member functionality across infected hosts for effects to determine member contribution to host range.2.ImportancePolydnaviruses are obligate mutualistic associates of highly speciose wasp taxa that parasitize caterpillars. Expression of Polydnavirus-encoded genes in hosts parasitized by wasps is necessary for successful parasitization, and an unusual genome structure including multiple-membered gene families is hypothesized to contribute to host manipulation. We have tested this hypothesis by in vivo expression of two members of a family of Polydnavirus homologues of Innexins, or insect gap junction proteins. Previous findings demonstrated that the two Vinnexins induce different physiological alterations in heterologous systems. Here, in host caterpillars, we observed differential alteration by the two proteins of host immune cell (hemocyte) bioelectrical physiology and the immune response of encapsulation. Not only do our data suggest a linkage between cellular bioelectricity and immunity in insects, but they support that gene family expansion has functional consequences to both Polydnavirus and host wasp success.

scholarly journals Particle Assembly and Ultrastructural Features Associated with Replication of the Lytic Archaeal Virus Sulfolobus Turreted Icosahedral Virus

2009 ◽  
Vol 83 (12) ◽  
pp. 5964-5970 ◽  
Author(s):  
Susan K. Brumfield ◽  
Alice C. Ortmann ◽  
Vincent Ruigrok ◽  
Peter Suci ◽  
Trevor Douglas ◽  
...  
Keyword(s):  
Time Course ◽  
Plaque Assay ◽  
Ultrastructural Changes ◽  
Progeny Virus ◽  
Particle Assembly ◽  
Virus Particles ◽  
Transmission Electron ◽  
Archaeal Viruses ◽  
Infected Cells ◽  
Sulfolobus Turreted Icosahedral Virus

ABSTRACT Little is known about the replication cycle of archaeal viruses. We have investigated the ultrastructural changes of Sulfolobus solfataricus P2 associated with infection by Sulfolobus turreted icosahedral virus (STIV). A time course of a near synchronous STIV infection was analyzed using both scanning and transmission electron microscopy. Assembly of STIV particles, including particles lacking DNA, was observed within cells, and fully assembled STIV particles were visible by 30 h postinfection (hpi). STIV was determined to be a lytic virus, causing cell disruption beginning at 30 hpi. Prior to cell lysis, virus infection resulted in the formation of pyramid-like projections from the cell surface. These projections, which have not been documented in any other host-virus system, appeared to be caused by the protrusion of the cell membrane beyond the bordering S-layer. These structures are thought to be sites at which progeny virus particles are released from infected cells. Based on these observations of lysis, a plaque assay was developed for STIV. From these studies we propose an overall assembly model for STIV.

Semliki Forest virus induced cell-cell fusion at neutral extracellular pH

1990 ◽  
Vol 10 (4) ◽  
pp. 363-374 ◽  
Author(s):  
Christoph Kempf ◽  
Marcel R. Michel ◽  
Adames Omar ◽  
Pia Jentsch ◽  
Andreas Morell
Keyword(s):  
Cell Fusion ◽  
Semliki Forest Virus ◽  
Covalent Modification ◽  
Surface Proteins ◽  
Acidic Ph ◽  
Fusion Event ◽  
Cell Surface Proteins ◽  
Infected Cells ◽  
Peptide Segments ◽  
Cell Cell

Semliki Forest virus-induced cell-cell fusion from within was considered to exclusively occur at mildly acidic pH (<6.2). Data of this study show that such cell fusion can also be triggered by transient acidification of the cytoplasm of infected cells at an extracellular, neutral pH. Results were obtained by utilizing NH4Cl pulses combined with covalent modification of cell surface proteins. The observation implies a revision of the current consensus regarding the mechanism of Semliki Forest virus induced cell-cell fusion. We propose a model in which at least two peptide segments of the viral spike protein E1 may be involved in triggering the fusion event.

scholarly journals Novel Immunofluorescence Assay Using Recombinant Nucleocapsid-Spike Fusion Protein as Antigen To Detect Antibodies against Severe Acute Respiratory Syndrome Coronavirus

2005 ◽  
Vol 12 (2) ◽  
pp. 321-328 ◽  
Author(s):  
Qigai He ◽  
Ivanus Manopo ◽  
Liqun Lu ◽  
Bernard P. Leung ◽  
Hiok Hee Chng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Fusion Protein ◽  
Autoimmune Diseases ◽  
Recombinant Baculovirus ◽  
Immunofluorescence Assay ◽  
Attractive Alternative ◽  
Sf9 Cells ◽  
Serum Samples ◽  
Detection Rates ◽  
Singapore General Hospital

ABSTRACT Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a “gold standard” during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.

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