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United States Department of Agriculture

Agricultural Research Service

Title: Ultrasonic Purification of Two Illinois Isolates of Barley Yellow Dwarf Viruses

Authors
item Latorre, Isabel - UNIV OF ILLINOIS
item Domier, Leslie
item D'Arcy, Cleora - UNIV OF ILLINOIS

Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 6, 1996
Publication Date: N/A

Interpretive Summary: Barley yellow dwarf viruses causes significant yield losses in cereal grains including oats, wheat barley and maize. The presence of the virus is most commonly determined using antibodies that have been produced against purified virus particles. However, purification of virus particles is difficult because the viruses accumulate only in those few cells that make up the vascular tissues of the plant. Because the vascular tissues have very strong cell walls, it is difficult break the cells open and release the virus particles during virus purification. Different methods have been tried to break up these tissues. One of the most successful has been the use of enzymes isolated from fungi that digest the plant cell walls. While the use of these digestive enzymes increase yields of virus particles, the virus preparations are often contaminated with other partially digested plant materials. To attempt to overcome these difficulties, we investigated the use of high intensity sound waves generated by an ultrasonic cell disrupter to break open the cells containing barley yellow dwarf virus particles. The yields of virus from sonicated tissue were about half of those obtained when using the digesting enzymes, but the virus preparations were of a much greater purity. These results will be useful to scientists who wish to produce diagnostic reagents to identify plants infected with barley yellow dwarf virus.

Technical Abstract: Ultrasonic disruption was used in the purification of two Illinois strains of barley yellow dwarf viruses (BYDV-PAV-IL and BYDV-RMV-IL). Purification yields as high as 366 mg/kg of BYDV-PAV-IL were obtained. The relative efficiencies of extraction of BYDV-PAV-IL with ultrasonic disruption, repeated blending and an industrial grade macerating enzyme (Extractase P20X) were compared. Use of sonication during extraction yielded an average of 1.5-fold more virus than repeated blending. Yields from sonicated tissue were half of those obtained with Extractase P20X but virus preparations were of a greater purity (as determined by their A260/A280 ratio). Sonication did not increase significantly yields of BYDV-RMV-IL when compared to repeated blending during extraction. However, the use of ultrasonic disruption in BYDV-PAV purification resulted in virus preparations that were sufficiently pure to be used as immunogens directly, and in a one-day decrease in the purification protocol compared to enzyme extraction.

Last Modified: 8/1/2014
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