Abstract：
Porcine astrovirus (PAstV) is a common cause of diarrhea in swine farms. The current understanding of the molecular virology and pathogenesis of PAstV is incomplete, especially due to the limited functional tools available. Here, ten sites in the open reading frame 1b (ORF1b) of the PAstV genome were determined to tolerate random 15 nt insertions based on the infectious full-length cDNA clones of PAstV using transposon-based insertion-mediated mutagenesis of three selected regions of the PAstV genome. Insertion of the commonly used Flag tag into seven of the ten insertion sites allowed the production of infectious viruses and allowed their recognition by specifically labeled monoclonal antibodies. Indirect immunofluorescence showed that the Flag-tagged ORF1b protein partially overlapped with the coat protein within the cytoplasm. An improved light-oxygen-voltage (iLOV) gene was also introduced into these seven sites, and only one viable recombinant virus that expressed the iLOV reporter gene at the B2 site was recovered. Biological analysis of the reporter viruses showed that these exhibited similar growth characteristics to the parental virus, but they produced fewer infectious virus particles and replicated at a slower rate. The recombinant viruses containing iLOV fused to ORF1b protein, which maintained their stability and displayed green fluorescence for up to three generations after passaging in cell culture. The porcine astroviruses (PAstVs) expressing iLOV were then used to assess the in vitro antiviral activities of mefloquine hydrochloride and ribavirin. Altogether, the recombinant PAstVs expressing iLOV can be used as a reporter virus tool for the screening of anti-PAstV drugs as well as the investigation of PAstV replication and the functional activities of proteins in living cells.
摘要：
猪星形病毒（PAstV）是猪场腹泻的常见原因。目前对PAstV的分子病毒学和发病机制的认识还不完全,特别是由于有限的功能工具。这里,使用基于转座子的插入介导的PAstV基因组三个选定区域的诱变，基于PAstV的感染性全长cDNA克隆，确定了PAstV基因组开放阅读框1b（ORF1b）中的10个位点可耐受随机15个nt插入。将常用的Flag标签插入10个插入位点中的7个允许产生感染性病毒并允许它们被特异性标记的单克隆抗体识别。间接免疫荧光显示Flag标记的ORF1b蛋白与细胞质内的外壳蛋白部分重叠。一个改进的光氧电压（iLOV）基因也被引入到这七个位点，并且仅回收一种在B2位点表达iLOV报告基因的活重组病毒。报告病毒的生物学分析表明，这些病毒表现出与亲本病毒相似的生长特征，但它们产生的感染性病毒颗粒较少，复制速度较慢。含有与ORF1b蛋白融合的iLOV的重组病毒，在细胞培养物中传代后，它们保持了稳定性并显示了多达三代的绿色荧光。然后使用表达iLOV的猪星形病毒(PAstV)来评估盐酸甲氟喹和利巴韦林的体外抗病毒活性。总之,表达iLOV的重组PAstV可用作报告病毒工具，用于筛选抗PAstV药物以及研究PAstV复制和活细胞中蛋白质的功能活性。
