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Identification and bio-control of viruses that causes diseases in humans, domestic animals and wild ruminants represent continuous challenges to human, agricultural and veterinary medicine. Thus, we plan to develop more effective bluetongue virus (BTV) approaches that can determine the mechanisms of viral infection and provide effective and protective means against the 24 known BTV serotypes. We will study the functions of the BTV non-structural protein NS-2 and its inhibitory effort on the production of infectious viral progeny. We also plan to utilize specific peptides representing its antigenic epitopes and ssRNA-binding domains to develop two unique kits that can differentiate whether an animal is vaccinated with BTV vaccine or infected by BTVs as well as a kit that can isolate only ssRNAs from cells and tissues.
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The following two major specific aims and objectives will be investigated: <OL> <LI> Functional definition of the BTV NS-2 protein including: (A) We will identify the amino acids critical and essential for both the antigenic epitopes and the ssRNA-specific binding regions; (B) We will determine the inhibitory mechanism(s) in the production of infectious viral progeny by excessive NS-2 protein expression; (C) We will use gene-silencing RNA inference (RNAi) for determination of gene functions and as potential therapeutics for Bluetongue disease. <LI> Biotechnological development of two kits with NS-2 functional peptides: (A) We will develop a magnetic bead-based kit for the specific isolation of ssRNAs from prokaryotic and eukaryotic cells. (B) We will also develop a peptide-based assay to differentiate a BTV-vaccinated animal from an animal infected with BTV.
NON-TECHNICAL SUMMARY: Bluetongue virus is a worldwide disease in domestic animal and wild ruminant and recently being considered as a bio-terrorist agent. DNA vaccine and the use of BTV NS-2 protein against this disease are being developed. This will contribute significantly to identify biologically active agents that can be used against BTV that bio-terrorists might use against US. NS-2 protein can also be used to develop two kits that can make definitive diagnosis of BTV-infected or vaccinated animals and specific isolation of all ss-RNAs for genomic investigation.
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APPROACH: (A) Identification of the critical amino acids critical and essential for both the antigenic epitopes and the ssRNA-specific binding domains: We will identify antigenic epitopes of NS2 of BTV using monoclonal, polyclonal, and oligoclonal antibodies that we have by immunoblotting, the minitope technique, competition with synthetic peptides, ELISA, immuno-precipitation, and the antigen capture assay. We will use the minitope technique to confirm the critical amino acid essential for these epitopes. (B) Determination of the inhibitory mechanism in the production of infectious viral progeny by excessive NS-2 protein. Excessive NS2 protein in the cells inhibited the production of infectious BTV17 by 10,000X. We plan to repeat this study using a new protein delivery system called the ChariotTM that provides uniform delivery of various amounts of intact and truncated NS2 proteins to different host cells at pre-determined times before infection with BTV. These will provide us with quantization and kinetics of the inhibition of viral progeny production. (C) Use of gene-silencing RNA interference (RNAi) for determination of gene function and as potential therapeutics for Bluetongue diseases. We will make siRNAs of 21 bp against the 10 BTV genes and use cell culture, transfections with lipofectamine, RNA isolation with RNeasy kit, PCR, RT-PCR, SDS-PAGE, 2-D gels, immunoblotting, ELISA with anti-BTV sera, plaque assay to determine inhibition of viral replication and the yield of BTV progeny. (D) Development of a magnetic bead-based kit for the specific isolation of ssRNAs from prokaryotic and eukaryotic cells. Since three synthetic peptides of NS-2 protein can bind ssRNAs only, we plan to develop a ssRNA-specific isolation kit by coupling 1-3 of these NS2 peptides either alone or in different combination to a magnetic bead to isolate all ssRNAs from cells. (E) Development of a peptide-based assay to differentiate a BTV-vaccinated animal from an animal infected with BTV. Sera of BTV-infected animals contain antibodies against all seven BTV structural proteins and three non-structural proteins. However, sera from animals vaccinated with non-infectious BTV vaccines have only antibodies against the structural but not the non-structural proteins. We will use synthetic peptides corresponding to the antigenic epitopes of VP7 and NS-2 proteins as testing substrates using the Synthetic Peptide Immuno-Blot Assay (SPIBA).
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PROGRESS: 2004/07 TO 2008/06 <BR>
OUTPUTS: The Synthetic Peptide ImmunoBlot Assay (SPIBE) that we have developed in the last 18 months has now been optimized and it can differentiate sheep and goat that are either infected with BTV or vaccinated with BTV vaccines from their serum samples. <BR> PARTICIPANTS: School of Medicine, Wuhan University, Wuhan, Chinax <BR> TARGET AUDIENCES: Bioagriculture and Diagnostics <BR> <BR>
IMPACT: 2004/07 TO 2008/06<BR>
The synthetic peptides representing the immunodominant and antigenic epitopes of non-structural protein NS-2 and the major viral protein VP7 of the Bluetongue virus can now be easily produced by the automatic peptide synthesizer at the Center of Integrated BioSystems at Utah State University. These peptides are quite stable at 4C or -20C for over 2 years with very minor degradation. We are confident that this synthetic peptide-based assay is a rapid diagnostic kit which can identify several BTV serotypes.