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. 2023 Jul 31;12(8):1001.
doi: 10.3390/pathogens12081001.

Bioreactor Production Process of Spodoptera frugiperda multiple nucleopolyhedrovirus Biopesticide

Karina Klafke  1 , Marcio Martinello Sanches  2 , William Sihler  3 , Marlinda Lobo de Souza  3 , Aldo Tonso  1
Affiliations

Bioreactor Production Process of Spodoptera frugiperda multiple nucleopolyhedrovirus Biopesticide

Karina Klafke et al. Pathogens. .

Abstract

Spodoptera frugiperda (fall armyworm) is one of the most important maize pests in the world and the baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), a natural pathogen of this pest, has been used as a biopesticide for its control. At present, in vivo strategies at the commercial scale are employed by multiplying the virus in the host insect in biofactory facilities; however, in vitro large-scale production is an interesting alternative to overcome the limitations of baculoviruses massal production. This study aimed to develop the process of the SfMNPV in vitro production by evaluating the effects of different multiplicities of infection (MOI) and nutritional supplements, morphological and molecular analysis of the infection on the growth of Sf9 cells and virus production. The Bioreactor Stirred Tank Reactor (STR) approach with glutamine-supplemented Sf-900 III serum free culture medium, combined with the MOI of 1.0, showed the best viral production performance, with a specific productivity above 300 occlusion bodies (OBs)/cell and volumetric productivity of 9.0 × 1011 OBs/L.

Keywords: SfMNPV; Spodoptera frugiperda; baculovirus; biological control; bioreactor; fall armyworm; in vitro production.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Kinetics of Run 1: Sf9 cell growth and metabolic analysis of glucose, lactate, glutamine, glutamate, and ammonium in STR bioreactor without glutamine supplementation. Samples were collected daily for cell-counting, expressed as viable cells ×106/mL. Glucose, glutamine, and lactate were quantified and expressed as g/L.
Figure 2
Figure 2
Kinetics of Run 2: Sf9 cell growth and metabolic analysis of glucose, lactate, glutamine, glutamate, and ammonium in STR bioreactor with glutamine supplementation at the end of the exponential growth phase. Samples were collected daily for cell-counting, expressed as viable cells ×106/mL. Glucose, glutamine, and lactate were quantified and expressed as g/L. The time axis was shifted to adjust the growth lag phase between runs.
Figure 3
Figure 3
Kinetics of Run 3: Sf9 cell growing in glutamine–supplemented bioreactor and infected with SfMNPV-19 at day 3.9 (MOI 0.1). Samples were collected for cell-counting, expressed as viable cells/mL, and viability (%). OBs were counted and expressed as OBs/mL. Cells with OBs were expressed as % of infected cells. The time axis was shifted to adjust the lag growth phase between runs. The vertical line represents infection time.
Figure 4
Figure 4
Kinetics of Run 4: Sf9 cell growth in glutamine-supplemented bioreactor and infected with SfMNPV-19 at day 3.8 (MOI 1.0). Samples were collected for cell-counting, expressed as viable cells/mL, and viability (%). OBs were counted and expressed as OBs/mL. Cells with OBs were expressed as % of infected cells. Viral DNA concentrations were expressed in ng/mL. The vertical line represents infection time.
Figure 5
Figure 5
Optical micrographs with magnification of 64× and 160× of SfMNPV infection progress in Sf9 cells cultivated in STR bioreactor. 0 h (before SfMNPV infection). 3, 4, 5, 6, 7 days post-SfMNPV infection (dpi). The arrows point to the OBs inside the nuclei, which indicate morphological alterations induced by the virus.
Figure 6
Figure 6
Electron micrographs of mock-infected Sf9 cells with 8000× magnification (A) and cells infected with SfMNPV-19 in STR bioreactor (B–D) at 7 dpi with 12,000×, 20,000× and 30,000× magnification, respectively. SEM performed with high voltage (HV) of 50,000 kV, horizontal field width (HFW) of 3.4 µm (B), 2.1 µm (C) and 1.4 µm (D).
See this image and copyright information in PMC

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