Author
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LYNN, DWIGHT |
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Submitted to: Journal of Methods of Cell Science
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/2/2002 Publication Date: 6/1/2002 Citation: Lynn, D.E. 2002. Effects of temperature on the susceptibility of insect cells to infection by baculoviruses. Journal of Methods of Cell Science. Interpretive Summary: Insect viruses are candidates for use as safe biopesticides for control of agricultural pests. Their production requires a living host cell, which can be living insects or cell cultures. To optimize production, an accurate estimate of the amount of virus needs to be determined. The current study addresses this problem by comparing the virus titers obtained din cell lines from three species: the fall armyworm, the gypsy moth and th tobacco budworm at three temperatures. In addition, three virus species were used: the alfalfa looper virus, the gypsy moth virus and the corn earworm virus. The first of these viruses is capable of infecting a wide range of insects and cell lines, while the latter two have a more restricted host range. Thus all three cell lines were tested with the alfalfa looper virus, but the gypsy moth virus was only tested on the gypsy moth cells and the corn earworm virus on the tobacco budworm cells. Results indicate the best temperature for accurate virus quantitation was 22 degrees C. The highest temperature tested showed 10 to 200-fold less virus on the fall armyworm and gypsy moth cells. Alternatively, the tobacco budworm cells showed little difference among the three temperatures, probably because this insect typically lives at higher temperatures. Other scientists or biopesticide manufacturers can use these results to accurately assay the amount of virus in their samples, a necessary goal for subsequent use of the material for optimizing production of these virus species. Technical Abstract: Three insect cell lines were tested for susceptibility to baculovirus infection by use of a typical endpoint assay procedure. Cell lines from Spodoptera frugiperda (IPLB-Sf21AE), Lymantria dispar (IPLB-LdEIta), and Heliothis virescens (IPLB-HvE6s) in 96-well tissue culture plates were each infected with dilutions of extracellular virus suspensions of the Autographa californica nucleopolyhedrovirus (AcMNPV). In addition, the L. dispar and H. virescens cells were also infected with L. dispar nucleopolyhedrovirus, and Helicoverpa zea nucleopolyhedrovirus, respectively. Each cell/virus combination was incubated at three temperatures: 22, 27 and 32C and wells were scored for positive infection (presence of occlusion bodies in cell nuclei) at two to four day intervals for up to four weeks. The resulting data were analyzed by the Spearman-Kerber method, providing virus titers for each combination of virus, cell line, and temperature. The results were categorized by accuracy (assuming the highest titer achieved was the most accurate) and by rapidity of maximum titer. AcMNPV reached the highest titer in each line at 22C although equivalent titers were reached with both AcMNPV and HzSNPV in the HvE6a line at all three temperatures. This line actually reported about 100-fold less AcMNPV than the other two lines with the same virus sample. Alternatively, the Sf21AE and LdEIta lines reached 10-fold higher titers at the lowest temperature as compared with the higher temperatures, although also at a slower rate. |
