|BIESBROCK, ANNA - University Of Texas|
|POWELL, CHRISTOPHER - University Of Texas|
|BEXTINE, BLAKE - University Of Texas|
Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/2014
Publication Date: 8/21/2014
Citation: Biesbrock, A.M., Powell, C.M., Hunter, W.B., Bextine, B.R. 2014. Propagation of Homalodisca Coagulata Virus-01 via Homalodisca Vitripennis cell culture. Journal of Visualized Experiments. 91. e51953-e51953
Interpretive Summary: An insect cell culture system was used to mass propagate a leafhopper-infecting virus with potential for use as a biological control agent. Cell cultures established from the glassy-winged sharpshooter leafhopper, GWSS. This insect is the primary vector for Pierce’s disease of grapevine in the USA. Pierce’s disease is caused by a plant-infecting bacterium which causes economic losses due to lost yields and plant deaths. A leafhopper virus discovered by ARS in 2005 was successfully mass produced in a cell culture system produced from the GWSS. This viral pathogen which is specific to leafhoppers may provide a new biological control agent to reduce the spread of disease pathogens by leafhoppers. The cell culture system may also be used to produce other insect viruses, such as those that infect ants and honey bees. This is a major advancement where cell lines do not yet exist for some insect species.
Technical Abstract: The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa, a xylem-limited bacterium that is the causal agent of Pierce's disease (PD) of grapevine. Pierce’s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells, HvWH, via embryonic tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.