Characterization of Novel Aptamers Specifically Directed to Red-Spotted Grouper Nervous Necrosis Virus (RGNNV)-Infected Cells for Mediating Targeted siRNA Delivery.

Lingli Zhou,Xiaohong Huang,Youhua Huang,Shina Wei,Qing Yu,Qiwei Qin,Jingguang Wei,Pengfei Li,Mingzhu Liu,Shaowen Wang

doi:10.3389/fmicb.2020.00660

Abstract

Nervous necrosis virus (NNV) causes viral nervous necrosis, the most devastating disease in more than 50 fish species worldwide, with massive mortality rates up to 100%, resulting in great economic losses to mariculture. However, few methods are available for the efficient diagnosis and treatment of viral nervous necrosis. Aptamers are molecular recognition ligands characterized by their remarkably high specificity and affinity, great stability, and ease of synthesis, and have been widely studied in application of disease diagnosis and therapies. In this study, we generated three aptamers against red-spotted grouper nervous necrosis virus (RGNNV)-infected grouper brain (GB) cells using the Cell-SELEX (cell based-systematic evolution of ligands by exponential enrichment) technology. The selected aptamers formed stable stem-loop structures, and could specifically recognize RGNNV-infected GB cells, with calculated dissociation constants (Kd) of 27.96, 29.3, and 59.5 nM for aptamers GBN2, GBN10, and GBN34, respectively. They also recognized RGNNV-infected brain tissues. The three aptamers were non-toxic and showed antiviral activities both in vitro and in vivo. Fluorescence microscopy and flow cytometry also demonstrated that aptamer GBN34 could be efficiently and specifically internalized into RGNNV-infected GB cells. The targeted cellular delivery of aptamer-small interfering RNA (siRNA) conjugates remarkably inhibited RGNNV infection in GB cells. The efficiency of the aptamer-based targeted delivery system was about 75% reduction in infection after 48 h, which was similar to that of transfection. These aptamers have great potential utility in the rapid diagnosis and inhibition of RGNNV infection in mariculture.

Highlights

Nervous necrosis virus (NNV), one of the most devastating marine viruses, typically causes viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in the larvae and juveniles of marine and freshwater fish (Souto et al, 2019)
The shift in the fluorescent signal showed that the 10th ssDNA pool bound most strongly to the RGNNVGB cells
Compared with the control cells incubated with red-spotted grouper nervous necrosis virus (RGNNV) alone, the relative CP mRNA expression decreased by 36.0% at 48 hp.i. in the cells exposed to mixtures of RGNNV and aptamer (100 nM), and by 75% in the cells exposed to mixtures of RGNNV and the aptamer–small interfering RNA (siRNA) conjugates (50 nM). These results demonstrate that the cellular delivery of the aptamer–siRNA conjugate contributed to the inhibition of RGNNV infection

Summary

Introduction

Nervous necrosis virus (NNV), one of the most devastating marine viruses, typically causes viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in the larvae and juveniles of marine and freshwater fish (Souto et al, 2019). RGNNV is the commonest pathogen associated with fish VNN diseases in Southeast Asian countries and China (Xu et al, 2010). Vacuolization of the brain and retinal tissues can be seen in infected fish, and common symptoms of VNN include abnormal whirling and spiraling, dark body color, and loss of appetite. Our knowledge of NNV infection and treatment is still limited, and there is no efficient commercial therapeutic regimen for VNN

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