Replication of a Nuclear Polyhedrosis Virus in a Continuous Cell Culture of
            <i>Spodoptera frugiperda:</i>
            Purification, Assay of Infectivity, and Growth Characteristics of the Virus

Knudson, D. L.; Tinsley, T. W.

doi:10.1128/jvi.14.4.934-944.1974

Cited by 93 publications

(35 citation statements)

References 16 publications

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“…This experiment confirmed our previous hypothesis that infected cells live longer in an oxygen-limited (low agitation) environment, even longer than uninfected cells when in an oxygenlimited stationary phase (see Figures 1 and 2). Since cell division and cellular DNA synthesis are blocked in infected cells, cellular regulation becomes controlled by the viral DNA replication, transcription, and translation activities (Fraser, 1989;Knudson and Tinsley, 1974). Consequently, if viral-associated activity is attenuated by the lack of oxygen, the cells remain in a suspended or quiescent state.…”

Section: Resultsmentioning

confidence: 99%

Effects of Oxygen/Glucose/Glutamine Feeding on Insect Cell Baculovirus Protein Expression: A Study on Epoxide Hydrolase Production

Wang

Kwong

Bentley

1993

Biotechnology Progress

101

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The recombinant protein yields for batch cultures of the insect cell baculovirus expression system have been significantly enhanced by oxygen, glucose, and glutamine feeding. The improvement in both volumetric and specific yields was based on influencing the metabolism of infected cells. Oxygen was absolutely required for viral replication and high protein expression in infected cells. Increases of 200% in volumetric yield and 100% in specific yield of recombinant epoxide hydrolase were achieved by controlling the dissolved oxygen (DO) level to near 35% saturation. An additional 100% increase was achieved by glucose and glutamine feeding. Results indicated that the intracellular metabolite pool was not adequate for recombinant protein overproduction. Finally, the specific protein yield, based on initial infection cell density, in high cell density spinner flasks and bioreactors of spent media with glucose and glutamine feeding was equivalent to that freshly diluted cultures.

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Section: Resultsmentioning

confidence: 99%

Effects of Oxygen/Glucose/Glutamine Feeding on Insect Cell Baculovirus Protein Expression: A Study on Epoxide Hydrolase Production

Wang

Kwong

Bentley

1993

Biotechnology Progress

101

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“…Of the cell culture and infection parameters that influence BEVS, the cell density at time of infection (TOI) has the most significant effect on the ability of insect cells to support virus replication and subsequent recombinant protein production. Very early literature indicated that the effect manifested itself as a significant decrease in volumetric production of both wild-type virus and occlusion bodies at high cell density infection (Hink et al, 1977;Knudson and Tinsley, 1974;Stockdale and Gardiner, 1977;Vaughn, 1976;Wood et al, 1982). The same limitation, however, has been observed to afflict the recombinant baculovirus expressed proteins and virus (Caron et al, 1990;Lindsay and Betenbaugh, 1992;Radford et al, 1991Radford et al, , 1992Reuveny et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Substrate limitation in the baculovirus expression vector system

Radford

Reid

Greenfield

1997

Biotechnol. Bioeng.

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The inability to infect insect cell cultures at the highest achievable cell densities has imposed major limitations to both the fundamental understanding of the Baculovirus Expression Vector System (BEVS) as well as full exploitation of its potential productive capacity for recombinant (β‐galAcNPV) products. The current literature does not characterize and identify the exact nature of the observed limitations, which therefore has become the major objective and contribution of the following study. Critical densities for infection of Spodoptera frugiperda (Sf9) cells with nuclear polyhedrosis virus expressing β‐galactosidase (Autographa californica) grown in media both containing fetal calf serum (FCS) and free of serum were found to be at 2 × 106 and 5 × 106 cells/ml respectively. Medium exchange was found to completely reverse the effect if renewed up to 24 hours post‐infection (HPI). The inevitable arrest of uninfected cell growth and decreased production of recombinant products at high cell densities of infection were both correlated to nutrient depletion. Cystine was found to be depleted in uninfected insect cell cultures at the onset of the stationary phase and in serum‐free insect cell cultures infected with baculovirus above a cell density of 5 × 106 cells/ml. Neither glucose depletion nor accumulation of possible inhibitory metabolites such as alanine, ammonia, or lactate could be correlated to growth arrest or decreased recombinant product yields. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 32–44, 1997.

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“…Methods have been developed for the gentle harvesting of insect cells using proteolytic enzymes. Because the replication and assay of NPV in cell culture is most efficient in low-density actively dividing cells [10][11][12][13] such harvesting methods can be of great use in studies on these viruses.…”

Section: Resultsmentioning

confidence: 99%

“…Because the replication of NPVs is best in actively dividing low-density cell monolayers [10][11][12][13], the quality of cells used for preparing such cultures is very important. The development of gentle methods using proteolytic enzymes for harvesting insect cells, analogous to those used with vertebrate cells [7], may therefore have considerable advantages for studying NPV replication in vitro.…”

Section: Introductionmentioning

confidence: 99%

The use of proteolytic enzymes for harvesting insect cell lines which support nuclear polyhedrosis virus replication

Roberts

1985

FEMS Microbiology Letters

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Various physical and enzymatic methods for harvesting insect cell lines, which grow as attached monolayers and which support the replication of nuclear polyhedrosis viruses (NPV), were investigated. Only 70 to 90% of cells were intact in cell suspensions produced by scraping and, on sub‐culture, the cells exhibited a lag phase of 1 to 2 days before growth resumed. An entirely satisfactory method for harvesting Spodoptera frugiperda cells using proteolytic enzymes could not be found. In contrast, trypsin or pronase could be used with Spodoptera littoralis cells. However, dispase, a neutral protease, proved to be the most effective enzyme giving 95 to 100% intact cells and, on sub‐culture, no inhibition of cell growth rate. Cells harvested using this gentle method are better for producing actively dividing low‐density cell monolayers which are needed for assaying and studying NPV.

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Replication of a Nuclear Polyhedrosis Virus in a Continuous Cell Culture of Spodoptera frugiperda: Purification, Assay of Infectivity, and Growth Characteristics of the Virus

Cited by 93 publications

References 16 publications

Effects of Oxygen/Glucose/Glutamine Feeding on Insect Cell Baculovirus Protein Expression: A Study on Epoxide Hydrolase Production

Effects of Oxygen/Glucose/Glutamine Feeding on Insect Cell Baculovirus Protein Expression: A Study on Epoxide Hydrolase Production

Substrate limitation in the baculovirus expression vector system

The use of proteolytic enzymes for harvesting insect cell lines which support nuclear polyhedrosis virus replication

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