title: Generation and characterization of novel DNA aptamers against coat protein of grouper nervous necrosis virus (GNNV) with antiviral activities and delivery potential in grouper cells.
abstract: Nervous necrosis virus (NNV) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. However, there is no efficient method to control NNV infections. Aptamers generated by selective evolution of ligands by exponential enrichment (SELEX) are short, single-stranded nucleic acid oligomers. They display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. In this study, three novel DNA aptamers (A5, A10, and B11) that specifically target the coat protein (CP) of grouper nervous necrosis virus (GNNV) were selected using SELEX. Secondary structures and minimum free energy (ΔG) predictions indicated that these aptamers could form stable, secondary stem-loop structures. Electrophoretic mobility shift assays, enzyme-linked immunosorbent assays, Kd measurements, the co-localization of tetramethylrhodamine (TAMRA) labeled-aptamers with the CP and flow cytometry analysis revealed that these aptamers could specifically bind the CP with high (nanomolar) affinities. In addition, competition analysis suggested the aptamers shared some common CP binding sites with the anti-CP antibody. Moreover, all three aptamers did not show any cytotoxic effects in vitro or in vivo, and anti-viral analysis indicated the selected aptamers could inhibit NNV infection in vitro and in vivo. Compared with controls, mortality of GNNV-infected fish decreased by 40% and 80% after 10 days infection, when the GNNV was pre-incubated with the 1000 nM A10 and B11, respectively. TAMRA-labeled aptamers could bind to NNV virions and directly enter NNV-infected cells, suggesting they could be used as tracers to study the mechanism of viral infection, as well as for targeted therapy. This is the first time that aptamers targeting a viral protein of marine fish have been generated and characterized. These aptamers hold promise as diagnostic, therapeutic, and targeted drug delivery agents for controlling NNV infections.
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China; University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing, 100049, China.
authors.forename: Lingli
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China; University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing, 100049, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Li
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China.
authors.forename: Min
authors.initials: M
authors.lastname: Yang
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China; University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing, 100049, China.
authors.forename: Yepin
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China.
authors.forename: Youhua
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China.
authors.forename: Jingguang
authors.initials: J
authors.lastname: Wei
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China. Electronic address: weisn@scsio.ac.cn.
authors.forename: Shina
authors.initials: S
authors.lastname: Wei
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, China. Electronic address: qinqw@scsio.ac.cn.
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Qin
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D036022:Capsid Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D005393:Fish Diseases / Q000188:drug therapy* / Q000821:virology; D005399:Fishes / Q000821:virology; D030381:Nodaviridae / Q000187:drug effects*; D012327:RNA Virus Infections / Q000188:drug therapy* / Q000821:virology; D014771:Virion / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D052157:Aptamers, Nucleotide; D036022:Capsid Proteins
keywords: Antiviral activity; Aptamer; Coat protein; Grouper nervous necrosis virus; SELEX