DEVELOPMENT OF RT PCR ASSAY FOR QUANTITATIVE DETECTION OF ENTERIC CALICIVIRUSES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 诺如病毒(NV)是重要的人类病原体，全球80%以上的急性胃肠炎暴发是由诺如病毒引起的。长期以来，由于缺乏有效的组织培养或动物模型，NVS的研究一直受到阻碍。最近我们报道了从恒河猴(Macaca Mulatta)(Farkas et al.)身上分离到的一种可培养杯状病毒(Tulane Virus；TV)，描述了一种代表杯状病毒科一新属的恒河猴杯状病毒。2008年；J·维罗尔。82(11)：5408-16)。根据初步结果显示TV和NV在遗传多样性、流行病学和组织血型抗原(HBGA)结合方面非常相似(未公布的数据)，以及反向遗传学系统的可用性(魏等人，通过转染体外产生的来自杜兰病毒cDNA的RNA来恢复传染性病毒)。2008年；J·维罗尔。82(22)：11429-36)，我们正在致力于开发一种基于电视的人类NV胃肠炎的非人类灵长类动物代理模型。为了在这项研究中支持这一努力，我们建立了一种定量RT-PCR方法，目的是通过恒河猴的受控感染过程来计数生物样本中的TV RNA载量。根据15株TV分离株的序列比对，设计了扩增依赖于RNA的RNA聚合酶的176个核苷酸的保守片段的引物。在扩增子内设计了针对原型(M33)TV的23个核苷酸长的寡核苷酸探针，并用报告荧光染料和荧光猝灭剂标记。这种基于Taq-man的检测方法对另外3个TV分离株具有很高的特异性，与目标区域的原型株有47%到82%的核苷酸同源性(基于质粒的检测)。该方法的最佳检测范围为5个对数，每个反应的检出限为10个TV基因组拷贝。从添加了组织培养原型TV的粪便样品中提取的TV基因组RNA也成功地被检测到。该检测方法可以对生物样品中的TV原型毒株进行高度特异和灵敏的定量，并将成为我们未来研究的重要工具。

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between TVs and NVs in respect to their genetic diversity, epidemiology and histo-blood group antigen (HBGA) binding (unpublished data) plus the availability of a reverse genetics system (Wei et al., Recovery of Infectious Virus by Transfection of in vitro Generated RNA from Tulane Virus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.