DEVELOPMENT OF RT PCR ASSAY FOR QUANTITATIVE DETECTION OF ENTERIC CALICIVIRUSES

Tulane University Of Louisiana, New Orleans LA

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Abstract

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. Noroviruses (NV) are important human pathogens that are responsible for over 80% of acute gastroenteritis outbreaks worldwide. Study of NVs is for long time hampered by the lack of an efficient tissue culture or animal model. Recently we reported the discovery of a cultivable calicivirus (Tulane virus;TV) isolated from a rhesus macaque (Macaca mulatta) (Farkas et al., Characterization of a rhesus monkey calicivirus representing a new genus of Caliciviridae. 2008;J Virol. 82(11):5408-16). Based on preliminary results that show close similarities between TVs and NVs in respect to their genetic diversity, epidemiology and histo-blood group antigen (HBGA) binding (unpublished data) plus the availability of a reverse genetics system (Wei et al., Recovery of Infectious Virus by Transfection of in vitro Generated RNA from Tulane Virus cDNA. 2008;J Virol. 82 (22):11429-36), 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 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.