Location:2010 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
Research over the past year has focused on defining the movement of purified virus though an aphid and discovering what proteins are associated with virus particles that may play a role in virus transmission. We found that plant proteins play a role in virus transmission, but that an endogenous bacteria aphid and the aphid hemolymph do not. Standard protocols for purification of luteoviruses use buffer containing EDTA, and sodium sulfite to preserve the infectivity of the virus by preventing oxidation and aggregation. While this is useful for many luteoviruses, it rendered preparations of purified CYDV-RPV non-transmissible. Viewed by EM, purified virions appeared normal in size and shape and reacted normally in serological tests. Protein analyses indicated that EDTA and sodium sulfite did not alter the virus protein composition, but they did affect the composition of host proteins that co-purify with the virus. The serendipitous discovery that virus purified using chelating and reducing agents lost transmissibility led to the discovery that specific plant host proteins may bind to the virus capsid and may play a regulatory role in virus recognition or movement through aphid vector tissues. This discovery broadens our perspective on the virus-plant host relationship and suggests that the coevolved partners may interact to effect virus transmission. Also we discovered an unknown bacteria present in the gut of an aphid genotype able to vector virus yet this bacteria was absent in the gut of an aphid (same species as the vector aphid) that cannot transmit virus. The bacteria was not transferred to any progeny and its presence was not correlated with transmission competence. Absence of bacterial infection in the progeny aphids indicates it is not paternally inherited and that the infection and the resulting cytopathology observed in the hindgut cells played no role in virus transmission. In fact, it is surprising that the severe cytopathology does not seem to significantly reduce or eliminate virus acquisition and suggests that aphid immune responses to thwart bacterial infections do not hinder virus acquisition in the gut. Another major discovery this year was that virus recovered from the hemolymph of nonvector aphids was transmissible when injected into vector competent aphids. Hemolymph from the nonvector aphids does not possess immunosuppressive effects on virus transmissibility or infectiousness. Virus isolated from the hemolymph of nonvector genotypes was equally infectious as that from vector genotypes. This eliminates the need for further emphasis on hemolymph factors on transmission and focuses future efforts on virus transport through the gut and salivary gland tissues as the main targets for disruption of transmission. Progress was monitored through regular lab meetings, email and phone conversations.