There are two components within this program. The first is to produce an ideal internal standard that resembles the target encapsulated viral particles being tested. In order to control all processing steps in molecular detection, this standard should have the same composition of target sequence and use the same primer set for amplification. This internal standard would be most valuable in assuring the validity of negative specimens during large-scale screening assays. The second component is to develop a new molecular amplification platform by combining two independent technologies using Mut-Y mismatch enzyme and TCR target cycling-based amplifications. These two technologies were brought together by a three-way CRADA. The end point for this collaboration is to develop a prototype test and to demonstrate its utility with clinical specimens. We have constructed a particulate HCV internal standard (IS) based on murine amphotropic retrovirus. To achieve this, we went through a stepwise process including mutating HCV genome by inserting a 36 nucleotide base at nt 272 position in the 5?-UTR and then creating a retroviral vector clone pXT-HCV-NCC-D8 containing 948 bases of the HCV sequence. This vector was used to transfect a retrovirus packaging cell line, PA317. From the transfected cells, G418 resistant recombinant retrovirus producer clones were established and further characterized. Using sequence specific primers, we were able to show that HCV sequence-containing particles were produced. Both wild type clones with HCV sequence and mutant clones with additional inserts were prepared and isolated. We were able to determine the insertion sequence length as expected by gene analyzer. One preparation of virus supernatant from a high virus producer, D8-54 was evaluated extensively. The relative copy number per milliliter was determined by different methods including RT/PCR titer, electron microscope particle counting, infectious colony-forming counts, and end-point infectious titer. We found consistent results with different methods of determination. This demonstrated that this approach could provide ideal particulate IS for HCV. We were also able to apply this IS to a small-scale study with clinical specimens. We were also able to develop a convenient EIA detection system based on insertion specificity. NIH filed a U.S. patent based on this work. For the second part of this project in finding new amplification approaches, in collaboration with Dr. Hsu of the University of Maryland, we found unique substrate specificity of Mut-Y enzyme that can recognize both DNA and RNA mismatches. The release mechanism for enzyme-substrate complex was determined. The cofactors, which enhance the turnover of substrate-product, were found. Potential target sequences on different strains of HIV were selected and specific probes were designed and synthesized. Ten- to thousand-fold amplification was demonstrated by estimating the probe products. Specificity was shown by a narrow range of strain specific recognition. A U.S. patent was filed and pending jointly by the University of Maryland and NIH based on these observations. To commercialize this patent, an industry partner capable of developing this technology was sought. Medical Analysis System of Camerillo, California presented the target cycle reaction (TCR) technology as an ideal partner. Combination between Mut-Y enzyme and TCR was shown to be compatible. High level of amplification was demonstrated. A U.S. patent based on the conditions set forth by the CRADA is being prepared for this combined technology.
