RECOVERY OF TULANE CALICIVIRUS BY TRANSFECTION OF IN VITRO GENERATED RNA

点击翻译按钮获取中文摘要

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.