DEVELOPMENT OF RT PCR ASSAY FOR QUANTITATIVE DETECTION OF ENTERIC CALICIVIRUSES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recently we reported the discovery of a cultivable calicivirus (Tulane virus; TV) isolated from a rhesus macaque. Based on preliminary results that show close similarities between TVs and noroviruses (NVs) in respect to their genetic diversity, epidemiology and histo-blood group antigen (HBGA) binding, plus the availability of in-house reverse genetics system, we are pursuing the development of a TV-based non-human primate surrogate model for human NV gastroenteritis. To support this effort in this study, we developed a qRT-PCR with the objective to enumerate TV RNA load in biological samples through the course of controlled infections of rhesus macaques. Based on alignments of 15 TV isolates, primers were designed to amplify a conserved 176 nucleotide fragment of the RNA dependent RNA polymerase. A 23 nucleotides long oligonucleotide probe specific for the prototype (M33) TV was designed within the amplicon and labeled with reporter fluorescent dye and fluorescence quencher. This TAQ-MAN based assay demonstrated a high level of specificity to the prototype TV against 3 other TV isolates with 47% to 82% nucleotide homology to the prototype strain in the target region (plasmid based assays). The assay allowed optimal detection in a 5-log range and a detection limit of 10 TV genomic copies per reaction. TV genomic RNA extracted from fecal samples spiked with tissue cultured prototype TV was also successfully detected. The assay described here allows for the highly specific and sensitive quantitation of the prototype TV strain in biological samples and will serve as an important tool for our future studies.