Caracterização molecular de mutantes gerados pela passagem serial do baculovírus Anticarsia gemmatalis MNPV em cultura de células

AUTOR(ES)

Syonara Hakiko Matusita Soares de Rezende

DATA DE PUBLICAÇÃO

2008

RESUMO

Baculovirus are insect viruses with great application potential for biological control programs due mainly to their ecological appeal, once they are highly specific to the host insect and do not present toxicity to man and to the environment. Their production has been made basically by their multiplication in host insects. Nevertheless, baculovirus production in cell cultures offers advantages over the in vivo multiplication for being a controllable, sterile, high pureness product yield process besides the fact of hundreds of cell lines being already established. However, successive passages of the virus in cell culture result in genetic alterations leading to loss of virulence. The most common alteration is the FP (Few Polyhedra) mutants formation due to mutations in the 25k fp gene. Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) baculovirus is being used successfully as control agent of the soybean caterpillar (A. gemmatalis). However, its production has been made in vivo and little is known on its production in cell cultures. In this work, the effects caused by the passage of the AgMNPV in BTI-Tn-5B1-4 cells was studied. The percentage of cells containing many polyhedra remained high and constant until the fifth passage. From this point on there was a significant reduction in the amount of cells with many polyhedra with concomitant increase in the amount of cells with few polyhedra. In parallel, there was a dramatic increase in the viral titer by production of extracellular virus (budded virus). All these parameters associated to the ultra structural analysis characterize the FP mutants generation during serial passage of the virus on cell culture. Additionally, there was similarity in the viral DNA restriction endonuclease digestion profile of different passages reveling no significant genetic modifications, such as deletions and insertions. The indication that small alterations, such as point mutations, present in the 25k fp gene locus was confirmed by this gene nucleic sequence determination in the selected mutants (seventh passage of supernatant). Point mutations in the 25k fp gene region were detected in three clones (FP1, FP3 and FP4), including its promoter region, and only one clone (FP5) did not present any alteration in this gene. Based on the protein synthesis kinetics using radioactive labeling, a reduction in the polhyedrin synthesis (the main component of the occlusion bodies) in all FP clones was also observed in comparison to the standard virus. Moreover, the ultra structural analysis of the FP clones disclosed typical characteristics of FP mutants formation such as cells with few or no polyhedra, amorphous polyhedra production with low virions number and virogenic stroma formation with intense nucleocapsid production. Finally, Many Polyhedra (MP) variants, presenting a larger number of polyhedra per cell and a similar to the parental virus budded virus titer, were selected as part of a strategy for a larger viral scale up production in cell culture (bioreactors).

ASSUNTO(S)

baulovírus mutantes fp gene 25 k fp produção in vitro passagem serial cultura de células biologia molecular

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