Location:2009 Annual Report
1a. Objectives (from AD-416)
Specific experiments to be completed at Penn State by Gildow and Cox-Foster include: l. Comparison of RPV subcellular localization in vector and non-vector genotypes utilizing immunofluorescent microscopy combined with immunogold labeling for ultrastructural identification of virus and virus-tissue interactions associated with transmission barriers. 2. Characterization of RPV trafficking pathways utilizing injection recovery bioassays from vector and nonvector aphids to verify acquisition of infectious virus and to study virus viability and stability in the aphid hemocoel environment.
1b. Approach (from AD-416)
Identification of RPV localization in S. graminum will use a tandem immunofluorescence (IF)-transmission electron microscopy (TEM) approach to facilitate viral subcellular localization by TEM. Briefly, aphids either membrane fed or microinjected with purified virus will be fixed and paraffin embedded. Paraffin sections of gut and salivary tissues will be incubated in primary virus-specific polyclonal antibody, followed by secondary antibody linked to Alexa fluor 488. These will pinpoint the cells for EM sectioning. Then routine ultrastructural TEM studies will determine the cellular mechanisms regulating virus recognition and transport. Aphids of each nonvector genotype will be examined for pathways of virus acquisition through various sites along the anterior and posterior midgut and hindgut. An additional exciting finding that might emerge from this study would be the identification of a hindgut or midgut escape barrier by which virus is permitted to enter and traffic through the cell, but is prevented from exiting. These results when combined with ultrastructural observations of virus associations with cellular organelles should indicate whether virions are moving along the endocytotic-exocytotic transmission pathway. Failure to transmit may be caused by structural barriers to diffusion through the basal plasmalemma of the gut or ASG, missing cellular components preventing virus recognition and endocytosis at the cell membranes, or disruption of the transcytosis pathway preventing directional movement of virions through gut or ASG cells. Virions accumulating in HG or ASG as a result of failure to be transported through cells are expected to be visualized as aggregates of virions in lysosomes. Characterization of RPV trafficking pathways will use injection recovery bioassays to characterize RPV acquisition ability of all vector and nonvector F2 clones and determine whether non-vector aphids with an apparent strong ASG barrier have an immunological response that degrades virions in the hemolymph. Aphids with infectious virions in the hemolymph, but unable to transmit will identify genotypes with a major ASG transmission barriers. Efficiency of virus recovery should parallel efficiency of virus acquisition. Virus acquisition in hemolymph could be quantitated by immunospecifc EM analysis and real-time RT-PCR if appropriate. Real-time RT-PCR methods to quantitate RPV in hemolymph were developed during the previous grant cycle. If RPV virions are visualized escaping from gut cells into the hemocoel by IF and TEM, and verified by RT-PCR, but infectious virus cannot be recovered by hemolymph bioassay, this would suggest an aphid cellular immunological basis for a loss of transmission. Such a discovery would open a new area of study, as virus viability in the aphid hemocoel has not been extensively examined.
3. Progress Report
Funding was not transferred to PSU until November 2008 and personnel to begin the research were not in place until January 2009. A majority of the effort has been in purifying the RPV strain of Cereal yellow dwarf virus for use in aphid transmission, virus localization and co-immunoprecipitation studies. Although virus can be purified from infected plant material in expected quantities and the structure of the virus appears normal when viewed under electron microscopy, the virus is not consistently infectious in the sense that it is not able to be inoculated to plants by the aphid vector, Schizaphis graminum. It was discovered that the addition of reducing agents and metal ion chelating agents during virus purification have an adverse affect on the infectivity of the virus, although they do not alter purification yield or gross virion structure. Noninfectious purified virus is detected in aphids fed on a solution of virus and sucrose, but virus is not observed in the hindgut of the aphid. Progress was monitored through regular lab meetings, email and phone conversations. Studies are underway to determine if the virus is not stable in the aphid or if it does not circulate normally through the aphid. Progress was monitored through regular lab meetings, email and phone conversations.