2013 Annual Report
1a.Objectives (from AD-416):
Specific objectives of this proposal are:.
1)Optimization of TriMV antiserum produced against in vitro expressed coat protein (CP) in E. coli using various forms of ELISAs;.
2)development of PCR and real-time reverse transcription (RT)-PCR detection methods for HPV; and.
3)development of real-time RT-PCR method for quantitative detection of WSMV and TriMV in wheat curl mites.
1b.Approach (from AD-416):
During 2010-11 funding cycle, we produced polyclonal antiserum against coat protein (CP) of TriMV. The CP was over expressed in E. coli, which was isolated as soluble form and used as an antigen for antiserum production in rabbits (see progress report). In preliminary experiments, we found that this antiserum is specifically reacting with TriMV in DAS-ELISA. Different batches (bleeds) of TriMV antiserum are available in our lab, and these antisera will be optimized using different forms of ELISAs such as direct antigen coating and double antibody sandwich to choose a suitable ELISA method for sensitive and high-throughput detection of TriMV.
We will perform partial genomic RNA sequencing from serologically distinct HPV isolates to obtain conserved stretches of sequences that can be used to design primers, and these primers will be used for PCR detection of HPV isolates from different regions of Nebraska. We will also utilize these conserved sequences in the genomes of HPV isolates to design primers and probe for real-time RT-PCR detection. The real-time PCR equipment is available in our lab.
The real-time RT-PCR method will be optimized for quantitative detection of WSMV and TriMV in wheat curl mites. By using this method, we can quantify the number of virus particles present in mite vectors, which can be used to find the minimum number of virus particles required in mites to be able to transmit the virus by mite vectors.
Efficient diagnostic methods are crucial for the management of viral diseases and for germplasm screening for virus resistance in breeding programs. Diagnostic methods for Triticum mosaic virus (TriMV), a recently found virus in the Great Plains region, are not available. Polyclonal antibodies were generated in rabbits against the bacterially expressed purified soluble native form of TriMV coat protein. In enzyme-linked immunosorbent assays (ELISA), antibodies reacted specifically beyond 1:20,000 dilution with TriMV in crude sap but not in extracts from healthy or WSMV-infected wheat. Notably, TriMV antibodies reacted positively with native virions in crude sap in a double antibody sandwich-ELISA, suggesting that these antibodies can be used as coating antibodies, which is crucial for any ‘sandwich’ type of assay for commercialization. Furthermore, the recombinant antibodies reacted positively in ELISA with representative TriMV isolates from the Great Plains region states, suggesting that TriMV antibodies can be used for sensitive, large-scale, and broad-spectrum detection. Furthermore, commercialization of these antibodies for the development of a TriMV diagnostic kit by a biotechnology company is in progress.
High plains virus (HPV) is a poorly studied, economically important virus in the Great Plains region. To develop more sensitive PCR-based diagnostic methods, two pairs of primers were designed from conserved regions of the nucleocapsid gene of available HPV isolates. These primers specifically amplified an expected DNA product from the total RNA of HPV-infected tissue but not from WSMV-infected or healthy tissue. These data suggest that HPV primers specifically detected HPV in wheat. Additionally, a real-time RT-PCR (Q-RT-PCR) method was developed for sensitive detection of HPV in wheat. The Q-RT-PCR method detected ~60 genomic RNA molecules of HPV and detected the virus in total RNA extracted from infected wheat leaves up to 1:1,280 dilution, which was several fold more sensitive than conventional PCR and ELISA detection methods. These sensitive detection methods will facilitate sensitive detection of HPV for the management of high plains disease in the Great Plains region.