DEVELOPMENT OF RT PCR ASSAY FOR QUANTITATIVE DETECTION OF ENTERIC CALICIVIRUSES

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 诺如病毒（NV）是重要的人类病原体，其负责全球超过80%的急性胃肠炎爆发。长期以来，由于缺乏有效的组织培养或动物模型，NVs的研究受到阻碍。最近，我们报道了从恒河猴（Macaca mulatta）分离的可培养杯状病毒（Tulane病毒; TV）的发现（Farkas等人，一种代表杯状病毒科新属的恒河猴杯状病毒的特征2008; J Virol. 82（11）：5408-16）。基于显示TV和NV在其遗传多样性、流行病学和组织血型抗原（HBGA）结合（未发表的数据）方面的密切相似性的初步结果，加上反向遗传学系统的可用性（Wei等人，通过从杜兰病毒cDNA转染体外产生的RNA回收感染性病毒。2008; J Virol. 82（22）：11429-36），我们正在开发用于人NV胃肠炎的基于TV的非人灵长类动物替代模型。为了支持本研究中的这一努力，我们开发了qRT-PCR，目的是通过恒河猴的受控感染过程来计数生物样品中的TV RNA负载。根据15个TV分离物的序列比对结果，设计引物，扩增了一个保守的176个核苷酸的RNA依赖的RNA聚合酶片段。在扩增子内设计了对原型（M33）TV特异的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。该基于TAQ-MAN的试验证明，原型TV对其他3种TV分离株具有高水平的特异性，与原型毒株在靶区域具有47%至82%的核苷酸同源性（基于质粒的试验）。该测定允许在5-log范围内进行最佳检测，并且每个反应的检测限为10个TV基因组拷贝。还成功地检测到从加入组织培养的原型TV的粪便样品中提取的TV基因组RNA。 该检测方法可以对生物样品中的原型TV株进行高度特异性和灵敏度的定量，并将作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between TVs and NVs in respect to their genetic diversity, epidemiology and histo-blood group antigen (HBGA) binding (unpublished data) plus the availability of a reverse genetics system (Wei et al., Recovery of Infectious Virus by Transfection of in vitro Generated RNA from Tulane Virus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.