A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line

Hopkins, Ralph F.; Esposito, Dominic

doi:10.2144/000113238

Cited by 94 publications

(80 citation statements)

References 14 publications

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“…These bioassays are also time-consuming, often requiring 4-7 days for the determination of the titer. Recently, some methods have been developed to reduce the time of titration, including the use of reporter genes such as b-galactosidase (LacZ) [9,10] and green fluorescent protein (GFP) [11,12], immunostaining with monoclonal antibodies against baculovirus proteins like surface glycoprotein 64 (gp64) [13] and the major capsid protein VP39 [14], direct staining of virus particles using SYBR Green I followed by flow cytometry (FCM) [15,16], quantitative real-time polymerase chain reaction (qPCR) [17,18], cell viability assay with AlamarBlue [19], and measurement of cell-diameter change of infected cells using a cell counter [20]. Nevertheless, these methods have several drawbacks which limit their widespread applications.…”

Section: Introductionmentioning

confidence: 99%

Rapid baculovirus titration assay based on viable cell side scatter (SSC)

Liu

Pan

et al. 2015

Analytica Chimica Acta

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Section: Introductionmentioning

confidence: 99%

Rapid baculovirus titration assay based on viable cell side scatter (SSC)

Liu

Pan

et al. 2015

Analytica Chimica Acta

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“…Spodoptera frugiperda (Sf21) insect cells (Invitrogen) were maintained as a monolayer culture in Grace's insect medium (Invitrogen), supplemented with 10% FBS (Hyclone). Sf9-easy titer (ET) cells (Hopkins and Esposito, 2009) were maintained as a monolayer culture in Sf-900 II (Invitrogen) serum-free medium. All cell cultures were supplemented with 50 U penicillin mL −1 , 50 g streptomycin mL −1 and 2 mM l-Glutamine (Gibco, Life Technologies).…”

Section: Cell and Virus Culturementioning

confidence: 99%

A sensitive epitope-blocking ELISA for the detection of Chikungunya virus-specific antibodies in patients

Goh

Kam

Metz

et al. 2015

Journal of Virological Methods

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“…Viral titers expressed as tissue culture infected dose 50 (TCID 50 ) per ml were determined by an end-point dilution assay (EPDA) using Sf9 Easy Titer cells (Hopkins and Esposito, 2009). Subsequent infections were performed by adding virus inoculum to the cells at a multiplicity of infection (MOI) of 10 TCID 50 units per cell.…”

Section: Cloning and Recombinant Bacmid Constructionmentioning

confidence: 99%

“…Viral titers were determined by an end-point dilution assay (EPDA) using Sf9 Easy Titration cells (Hopkins and Esposito, 2009) for baculoviruses or CHSE-214 cells for SAV. CHSE-214 cells were transfected at 12°C for 4 h using FuGene (Promega) according to manufacturer's protocol, then placed at 12°C or 20°C.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Recombinant baculoviruses were titrated using Sf9 'easy titration' cells (Hopkins and Esposito, 2009) and the titer was determined as tissue culture infectious dose 50 (TCID 50 ) per mL. Infection of Sf cells with recombinant baculovirus was performed at a multiplicity of infection (MOI) of 10 TCID 50 units per cell.…”

Section: Cell Culture Maintenance Transfection and Infectionmentioning

confidence: 99%

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Next-generation salmonid alphavirus vaccine development

Hikke¹

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Aquaculture is essential to meet the current and future demands for seafood to feed the world population. Atlantic salmon and rainbow trout are two of the most cultured aquaculture species. A pathogen that threatens these species is salmonid alphavirus (SAV). A current inactivated virus vaccine against SAV provides cross-protection against all SAV subtypes in salmonids and reduces mortality amongst infected fish. However, protection is not 100% and due to virus growth at low temperature, the vaccine production process is time consuming. In addition, the vaccine needs to be injected into the fish, which is a cumbersome process. The work described in this thesis aimed to increase the general knowledge of SAV and to assess current vaccine technologies, and to use this knowledge in designing next-generation vaccines for salmonid aquaculture.An alternative cell line to support SAV proliferation was identified, however, the virus production time could not yet outcompete the current SAV production system. Making use of the baculovirus insect cell expression system, multiple enveloped virus-like particle (eVLP), and core-like particle (CLP) prototype vaccines were produced in insect cells at high temperature. An in vivo vaccination study showed, however, that these vaccines could not readily protect Atlantic salmon against SAV. The low temperaturedependent replication of SAV was attributed to the glycoprotein E2, and it was found that E2 only correctly travelled to the cell surface at low temperature, and in the presence of glycoprotein E1. The biological impact of this finding was confirmed in the development and in vivo testing of a DNA-launched replicon vaccine. The effective DNAlaunched replicon vaccine was extended by delivery of the capsid protein in trans. It was hypothesized that viral replicon particles (VRP) were formed in vivo, which would cause an additional single round of infection and might further elevate the immune response in comparison to the replicon vaccine. A second animal trial indicated that the inclusion of capsid did not yet improve vaccine efficacy. This trial however did show that a DNA vaccine transiently expressing the SAV structural proteins provided superior protection over both replicon vaccines (with and without capsid).In this thesis, some virus characteristics, such as the cause of temperature-dependency of SAV replication, of an unique aquatic virus were further explored. The production and in vivo testing of multiple next-generation vaccines defined the prerequisites for induction of a potent immune response in Atlantic salmon. A prototype DNA-launched replicon vaccine has shown potential for further development. The research described in this thesis contributes to the development of next-generation vaccines in the challenging area of fish vaccinology. (European Commission, 2015). Aquaculture is indispensable, as wild-catch of seafood cannot meet the growing human consumption demands. In addition, aquaculture may allow the recovery of those wild fish stocks that have been ...

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A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line

Cited by 94 publications

References 14 publications

Rapid baculovirus titration assay based on viable cell side scatter (SSC)

Rapid baculovirus titration assay based on viable cell side scatter (SSC)

A sensitive epitope-blocking ELISA for the detection of Chikungunya virus-specific antibodies in patients

Next-generation salmonid alphavirus vaccine development

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