title: Rana grylio virus TK and DUT gene locus could be simultaneously used for foreign gene expression.
abstract: Ranaviruses (family Iridoviridae, genus Ranavirus) have been recognized as emerging infectious pathogens and caused a great loss to the global biodiversity. Thymidine kinase (TK) and deoxyuridine triphosphatase (dUTPase, DUT, encoded by ORF 67R) are ubiquitous, existing in iridoviruses and other organisms. Previous studies showed that TK and DUT could be individually knocked out without impeding viral replication. In this study, we tried to insert two fluorescence genes into the above loci. We started with Δ67R-RGV, a recently generated recombinant Rana grylio virus (RGV) with the whole DUT replaced by enhanced green fluorescence protein (EGFP) gene. Then, a red fluorescence protein (RFP) gene initiated by RGV immediate-early (IE) ICP18 gene promoter was inserted into TK locus through homologous recombination. A novel recombinant virus, ΔDUT, TK-RGV, was generated by nine successive rounds of plaque isolation using RFP selection. All of the plaques produced by this recombinant virus could emit both green and red fluorescence. Furthermore, one-step and multiple-step growth curves of ΔDUT, TK-RGV were similar to those of wt-RGV and Δ67R-RGV. In conclusion, a novel dual-fluorescence labeled recombinant iridovirus in which DUT and TK gene locus were simultaneously used for foreign gene expression was constructed.
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Fang
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan 430072, China. Electronic address: zhangqy@ihb.ac.cn.
authors.forename: Qiya
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D005818:Genetic Engineering; D056426:Genetic Loci; D010957:Plasmids / Q000235:genetics; D011755:Pyrophosphatases / Q000235:genetics*; D017995:Ranavirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013937:Thymidine Kinase / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D014764:Viral Proteins; D013937:Thymidine Kinase; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase
keywords: DUT); Deoxyuridine triphosphatase (dUTPase; Dual-fluorescence; Rana grylio virus (RGV); Recombinant iridovirus; Thymidime kinase (TK)