Identification Of Lepidopteran Insect Cell Lines By DNA Amplification Fingerprinting And Susceptibilities To Infection By SfaMNPV

Complete characterization of cell lines is essential for the cell banks and for the regulatory requirements in biopharmaceutical production.Characterization of a cell line provides information regarding the lineage or speciation of the cell line,genetic stability of cells,and cross-contamination of cell lines.Cell line characterization procedures classically include analysis of distinguishing markers of the cells.Insect cell lines are being widely used in research and industry for cultivation of insect viruses and production of recombinant proteins.Compared to the mammalian cell lines,the extent of cross-contamination and mislabeling is reported higher in cases of insect cell lines.The overall goal of this work was the identification of 17 lepidopteran insect cell lines by DNA Amplification fingerprinting and test susceptibility of insect cell lines to infection by syngrapha falcifera multiple nuclear polyhedrosis viruses(SfaMNPV).DNA amplification fingerprinting(DAF) with arbitrarily selected primers was used to obtain DNA fingerprint profiles to distinguish among 17 lepidopteran insect cell lines.The fingerprinting pattern is a stable characteristic of the cell line because high and low passages generated the same profile.The DNA from each cell line was amplified,and PCR products were analyzed by agarose gel electrophoresis.All cell lines could be distinguished from each other with following exception:BCIRL-HA-AM1 established from Heliothis armigera produced the same profile as Laphygma exigua(Le-H-HNU7). This case of identical patterns is believed to be due to cross contamination of cultures.Of the total 17 cell lines,six cell lines has been tested for susceptibility to Syngrapha falcifera multiple nucleocapsid nucleopolyhedrovirus(SfaMNPV) infection by use of a typical endpoint assay procedure.Cell lines from Trichoplusia ni BTITN5Bl-4 (TnH 5-S),Spodoptera frugiperda(Sf9 BCIRL clone),Spodoptera litura(SLZSU-1), Plutella xylostella BCIRL-PX2-HNU3,Lymantria dispar IPLB-Ld652,and Helicoverpa zea(BCIRL-HZ-AM1) in 96-well tissue culture plates were infected with dilutions of virus suspensions(SfaMNPV).Each cell/virus combination was incubated at temperatures 27℃and Cells were examined for cytopathic effect(the presence of occlusion bodies in the nuclei) from 2 to 4 days intervals up to one week post infection. The resulting data were analyzed by Reed and Muench method,providing virus titers for each combination of viral infection cell line.The results were categorized by accuracy and by rapidity of maximum titer.Virus titer of Sf9 BCIRL clone was higher than other cell lines 5×10⁷ TCID₅₀/ml,3.5×10⁷ pfu/mL the lowest level detected in infected cells was in BTI-TN5Bl-4(TnH 5-S) cells 3.2×10⁷ TCID₅₀/ml,2.2×10⁷ pfu/ml.No virions or polyhedral inclusion bodies were detected in SL-ZSU-1,BCIRL-HZ-AM1,IPLB-Ld652 and BCIRL-PX2-HNU3 cells.