Large-scale propagation of Homalodisca coagulata virus-1 via glassy-winged sharpshooter (Homalodisca vitripennis) tissue cell culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is an invasive insect species that has been identified throughout the southwestern United States.  Homalodisca vitripennis are highly vagile and polyphagous, feeding on more than 100 plant species.  They are the primary vector of Xylella fastidiosa (Xf), a xylem-limited bacterium that is the causal agent of Pierce's disease (PD) in grapevines.  Infected H. vitripennis transmit this bacterium during feeding, and as voracious xylem feeders are capable of rapidly infecting vineyards.  This disease has been economically damaging in agriculture and H. vitripennis have become a target for disease control.  A dicistroviridae virus known as Homalodisca coagulata virus -1 (HoCV-1) has been associated with an increase in mortality rates within infected H. vitripennis populations.  A host is required to replicate the virus and live insect rearing can present complications, making cell culture an approach that is logistically and economically valuable for mass-producing a biological control agent.  In this study, we developed a system for large-scale propagation of H. vitripennis cells via tissue culture, providing an ample supply of viral replication machinery.   Cells were inoculated with low levels of HoCV-1 and viral titers were taken for RNA extraction using TRIzol to determine optimal whole virus extraction times.  We were able to extract and purify whole virus particles within 72-96 hours post-inoculation before total cell culture collapse occurred. This study shows that H. vitripennis cells are capable of being cultured and used as viral replication machinery to produce a biological control agent.