Reduction of the infectivity of baculovirus stocks frozen at ultra-low temperature in serum-free media: The role of lipid emulsions.

The infectivity of stocks of baculoviruses produced in serum-free media is sensitive to freezing at ultra-low temperatures. The objective of this work was to elucidate the causes of such sensitivity, using as a model the freezing of stocks of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), a baculovirus widely employed as biological insecticide. Titers of supernatants of cell cultures infected with AgMNPV in four different serum-free media supplemented with lipid emulsions were reduced by 50 to 90% after six months freezing. By using a full factorial experiment, freezing and lipid emulsion, as well as the interaction between them, were identified as the main factors reducing the viral titer. The virucidal effect of the lipid emulsion was reproduced by one of their components, the surfactant Polysorbate 80. Damaged viral envelopes were observed by transmission electron microscopy in most particles frozen in a medium supplemented with lipid emulsion or Polysorbate 80. Additionally, Polysorbate 80 also affected the infectivity of AgMNPV stocks that were incubated at 27°C. The identification of the roles played by the lipid emulsion and Polysorbate 80 is not only a contribution to the understanding of the mechanisms underlying the inactivation of baculovirus stocks produced in serum-free media during storage at ultra-low temperature, but is also an input for the rational development of new procedures aimed at improving both the preservation of baculovirus stocks and the composition of culture media for the production of baculovirus-based bioproducts in insect cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1559-1569, 2016.

Production of the Anticarsia gemmatalis multiple nucleopolyhedrovirus in serum-free suspension cultures of the saUFL-AG-286 cell line in stirred reactor and airlift reactor.

The velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), is one of the main plagues for soybean crops. Velvetbean caterpillar larvae are susceptible to be infected by occlusion bodies of the baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), a biological insecticide. The insect cell line saUFL-AG-286 produces very high yields of occlusion bodies of AgMNPV in suspension cultures done in the low-cost serum-free medium UNL-10 in shake-flasks. However, its ability to adapt to conditions of industrial production in bioreactors was unknown. The aim of this study was to characterize the growth of saUFL-AG-286 cell cultures in UNL-10 medium, as well as its capability to replicate AgMNPV in two different bio-reactors at laboratory scale. The cell line was able to adapt to conditions that can be used at industrial scale, both in an airlift reactor and a stirred reactor, although the former was better than the last to support the cell growth. The infection with AgMNPV in the airlift reactor produced a high yield of occlusion bodies, with very low production of budded virus, the progeny used as inoculums. On the other hand, infection in the stirred reactor yielded high titers of budded virus. These results suggest that a feasible strategy for scaling-up the production of AgMNPV might involve the use of airlift reactors for the scaling-up of cell suspension cultures and the final production of occlusion bodies, while the scaling-up of the viral inoculums being carried out under conditions as those existing in stirred reactors.