Laboratory of Animal infectious Diseases and molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning 530005, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning 530005, China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning 530005, China.
Laboratory of Animal infectious Diseases and molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning 530005, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning 530005, China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning 530005, China.
Vet Microbiol. 2023 May;280:109675. doi: 10.1016/j.vetmic.2023.109675. Epub 2023 Feb 10.
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.
猪星状病毒（PAstV）是猪场内腹泻的常见原因。目前对 PAstV 的分子病毒学和发病机制的了解并不完整，特别是由于可用的功能工具有限。在这里，基于 PAstV 的感染性全长 cDNA 克隆，通过转座子插入介导的 PAstV 基因组三个选定区域的诱变，确定了 ORF1b 中的十个位点可耐受随机 15 个核苷酸插入。在这十个插入位点中的七个中插入常用的 Flag 标记，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。还将改进的光氧电压（iLOV）基因引入这七个位点，并且仅回收在 B2 位点表达 iLOV 报告基因的一个可行重组病毒。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 iLOV 融合到 ORF1b 蛋白的重组病毒在细胞培养中传代后，其稳定性得以维持，并且可显示绿色荧光长达三代。然后，使用表达 iLOV 的猪星状病毒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 可作为筛选抗 PAstV 药物以及研究 PAstV 复制和蛋白质在活细胞中的功能活性的报告病毒工具。
