Decline in Helicoverpa armigera nucleopolyhedrovirus occlusion body yields with increasing infection cell density in vitro is strongly correlated with viral DNA levels.

The phenomenon of the reduction in the cell-specific yield with increasing infection cell density (ICD), the cell density effect, is one of the main hurdles for improving virus yields in vitro. In the current study, the reduction in cell-specific yields (viral DNA [vDNA], polyhedrin mRNA and occlusion body [OB]) with increasing ICD for Helicoverpa armigera nucleopolyhedrovirus (HearNPV)-infected HzAM1 (Helicoverpa zea) insect cells has been investigated. HzAM1 cells were propagated in Sf900™ III serum-free medium and synchronously infected with wild-type HearNPV at various ICDs of 0.

Effect of the peak cell density of recombinant AcMNPV-infected Hi5 cells on baculovirus yields.

The phenomenon of the cell density effect is not readily explained by an obvious nutrient limitation, and a recent study has suggested that for recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV)-infected Sf9 cells, a drop in messenger RNA (mRNA) levels may be sufficient to explain the cell density effect for this system. The current study aims to investigate the response in cell-specific yields (viral DNA (vDNA), LacZ mRNA and β-galactosidase (β-Gal) protein) with increasing infection cell density (ICD) for rAcMNPV-infected Hi5 cells, where the rAcMNPV expresses the β-Gal gene under control of the polyhedral promoter. Hi5 cells in suspension culture of Express Five® medium were synchronously infected with a rAcMNPV at multiple ICDs between 0.

Decline in baculovirus-expressed recombinant protein production with increasing cell density is strongly correlated to impairment of virus replication and mRNA expression.

The cell density effect is a well-established constraint in the baculovirus-insect cell expression platform, in which cell-specific productivity declines with increasing cell density, hence limiting the maximum achievable volumetric yield of protein product. A deeper elucidation of this phenomenon is sought in this study, by tracking the peak production of viral DNA (vDNA), recombinant LacZ mRNA, and β-galactosidase (β-gal) protein, over a wide range of cell densities. Sf9 suspension cell cultures were propagated in Sf-900 III serum-free medium and synchronously infected with rAcMNPV at multiple infection cell densities (ICDs) of between 0.

Improving the robustness of a low-cost insect cell medium for baculovirus biopesticides production, via hydrolysate streamlining using a tube bioreactor-based statistical optimization routine.

A critical component of an in vitro production process for baculovirus biopesticides is a growth medium that is efficacious, robust, and inexpensive. An in-house low-cost serum-free medium, VPM3, has been shown to be very promising in supporting Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) production in H. zea insect cell suspension cultures, for use as a biopesticide against the Heliothine pest complex.

Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells.

Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility.