RECOVERY OF TULANE CALICIVIRUS BY TRANSFECTION OF IN VITRO GENERATED RNA

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。 我们最近从恒河猴的粪便中分离到一种新的杯状病毒，命名为杜兰病毒(TV)。该病毒已成功适应于细胞培养，并在恒河猴肾细胞(LLC-MK2)中引起典型的溶细胞性感染。TV的完整基因组序列表明，它代表了一个新的杯状病毒属，与人类诺如病毒有关。在这项研究中，我们证明了从克隆的cDNA体外转录的TV基因组RNA在LLC-MK2细胞中具有感染性。用T7RNA聚合酶体外转录得到病毒的全长RNA。将该RNA导入LLC-MK2细胞，细胞死亡呈剂量依赖性。RT-PCR检测病毒的RNA复制，细胞死亡和RNA复制的感染性病毒可连续传代，在1-2代内达到与组织培养适应病毒相似的病毒滴度。相反，未封顶的TV RNA或封顶的TV RNA在ORF1的上游插入EGFP的框内不会引起细胞病变(CPE)或RNA复制。从转染的RNA中也回收到含有一个引入突变标记的感染性TV。组织培养的可用性和我们新的反向遗传学系统使TV成为研究杯状病毒致病和复制的有价值的模型。

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. We recently isolated a novel calicivirus named Tulane virus (TV) from stools of rhesus monkeys. This virus has been successfully adapted in cell culture and causes typical cytolytic infection in rhesus monkey kidney cells (LLC-MK2). The complete genomic sequence of the TV suggested that it represents a new calicivirus genus that is related to human noroviruses. In this study, we demonstrated that in vitro transcribed TV genomic RNA from cloned cDNA is infectious in LLC-MK2 cells. Capped full-length viral RNA was generated by in vitro transcription with T7 RNA polymerase. Transfection of this RNA into LLC-MK2 cells resulted in cell death in a dose dependent manner. Viral RNA replication as monitored by RT-PCR, and infectious viruses as the cell death and RNA replication could be serially passed, reaching similar viral titers within 1-2 passages as that of the tissue culture-adapted virus. In contrast, uncapped TV RNA or capped TV RNA with an in-frame insertion of eGFP to the upstream of ORF1 did not cause cytopathic effect (CPE) or RNA replication. Infectious TV containing an introduced mutation marker was also recovered from transfected RNA. The availability of a tissue culture and our novel reverse genetics system makes TV a valuable model for studying calicivirus pathogenesis and replication.