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Characterization of DNA aptamers generated against the soft-shelled turtle iridovirus with antiviral effects.

Li P, Zhou L, Yu Y, Yang M, Ni S, Wei S, Qin Q - BMC Vet. Res. (2015)

Bottom Line: Soft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis).Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV.Aptamer QA-36 had the highest calculated binding affinity (K d ) of 53.8 nM.

View Article: PubMed Central - PubMed

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.

ABSTRACT

Background: Soft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis). More efficient methods of controlling and detecting STIV infections are urgently needed. 

Methods: In this study, we generated eight single-stranded DNA (ssDNA) aptamers against STIV using systematic evolution of ligands by exponential enrichment (SELEX).

Results: The aptamers formed representative stem-loop secondary structures. Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV. Aptamer QA-36 had the highest calculated binding affinity (K d ) of 53.8 nM. Flow cytometry and fluorescence microscopy of cell-aptamer interactions demonstrated that QA-12 was able to recognize both STIV-infected cells and tissues with a high level of specificity. Moreover, the selected aptamers inhibited STIV infection in vitro and in vivo, with aptamer QA-36 demonstrating the greatest protective effect against STIV and inhibiting STIV infection in a dose-dependent manner.

Discussion: We generated DNA aptamers that bound STIV with a high level of specificity, providing an alternative means for investigating STIV pathogenesis, drug development, and medical therapies for STIV infection.

Conclusions: These DNA aptamers may thus be suitable antiviral candidates for the control of STIV infections.

No MeSH data available.


Related in: MedlinePlus

Some selected aptamers bound STIV-infected FHM cells with a high level of specificity. a Fluorescence intensities of four selected FITC-aptamers binding to infected FHM cells. The fluorescence intensity of QA-12 was increased in STIV-infected FHM cells compared with QA-9, QA-36 and QA-92. The FITC-labeled initial library incubated with STIV-infected FHM cells were used as the control (black). b Fluorescent images of four FITC-aptamers with STIV-infected FHM cells. QA-12 bound to STIV-infected cells but not to normal cells. QA-9, QA-36, QA-92 and the library bound to neither STIV-infected cells nor normal cells. The initial library incubated with infected cells and aptamers incubated with noamal cells were used as the negative controls. c Fluorescent images of four FITC-aptamers with STIV-infected turtle tissues. QA-12 bound to STIV-infected liver and spleen tissues but not to normal tissues. QA-9, QA-36 and QA-92 bound to neither STIV-infected tissues nor normal tissues. The initial library was used as a negative control. Left: bright field; right: fluorescent. (Bars = 20 μm)
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Fig7: Some selected aptamers bound STIV-infected FHM cells with a high level of specificity. a Fluorescence intensities of four selected FITC-aptamers binding to infected FHM cells. The fluorescence intensity of QA-12 was increased in STIV-infected FHM cells compared with QA-9, QA-36 and QA-92. The FITC-labeled initial library incubated with STIV-infected FHM cells were used as the control (black). b Fluorescent images of four FITC-aptamers with STIV-infected FHM cells. QA-12 bound to STIV-infected cells but not to normal cells. QA-9, QA-36, QA-92 and the library bound to neither STIV-infected cells nor normal cells. The initial library incubated with infected cells and aptamers incubated with noamal cells were used as the negative controls. c Fluorescent images of four FITC-aptamers with STIV-infected turtle tissues. QA-12 bound to STIV-infected liver and spleen tissues but not to normal tissues. QA-9, QA-36 and QA-92 bound to neither STIV-infected tissues nor normal tissues. The initial library was used as a negative control. Left: bright field; right: fluorescent. (Bars = 20 μm)

Mentions: To determine if aptamers against STIV recognized STIV-infected cells, we incubated FITC-aptamers (300 nM) with STIV-infected FHM cells and monitored the process by flow cytometry. The FITC-labeled initial library incubated with STIV-infected FHM cells were used as the control. Compared to the control, the fluorescence intensity of QA-12 increased in STIV-infected FHM cells, though the effects of QA-9, QA-36 and QA-92 were less noticeable, which means only QA-12 bound to STIV-infected cells, while QA-9, QA-36, QA-92 and the library could not bind to STIV-infected cells (Fig. 7a). These results were verified by imaging infected cell-aptamer interactions (Fig. 7b). Aptamer QA-12 evolved against STIV could thus recognize STIV-infected FHM cells. Fluorescent images of liver and spleen tissues indicated that the selected aptamers bound STIV-infected liver and spleen tissues with high levels of specificity (Fig. 7c).Fig. 7


Characterization of DNA aptamers generated against the soft-shelled turtle iridovirus with antiviral effects.

Li P, Zhou L, Yu Y, Yang M, Ni S, Wei S, Qin Q - BMC Vet. Res. (2015)

Some selected aptamers bound STIV-infected FHM cells with a high level of specificity. a Fluorescence intensities of four selected FITC-aptamers binding to infected FHM cells. The fluorescence intensity of QA-12 was increased in STIV-infected FHM cells compared with QA-9, QA-36 and QA-92. The FITC-labeled initial library incubated with STIV-infected FHM cells were used as the control (black). b Fluorescent images of four FITC-aptamers with STIV-infected FHM cells. QA-12 bound to STIV-infected cells but not to normal cells. QA-9, QA-36, QA-92 and the library bound to neither STIV-infected cells nor normal cells. The initial library incubated with infected cells and aptamers incubated with noamal cells were used as the negative controls. c Fluorescent images of four FITC-aptamers with STIV-infected turtle tissues. QA-12 bound to STIV-infected liver and spleen tissues but not to normal tissues. QA-9, QA-36 and QA-92 bound to neither STIV-infected tissues nor normal tissues. The initial library was used as a negative control. Left: bright field; right: fluorescent. (Bars = 20 μm)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig7: Some selected aptamers bound STIV-infected FHM cells with a high level of specificity. a Fluorescence intensities of four selected FITC-aptamers binding to infected FHM cells. The fluorescence intensity of QA-12 was increased in STIV-infected FHM cells compared with QA-9, QA-36 and QA-92. The FITC-labeled initial library incubated with STIV-infected FHM cells were used as the control (black). b Fluorescent images of four FITC-aptamers with STIV-infected FHM cells. QA-12 bound to STIV-infected cells but not to normal cells. QA-9, QA-36, QA-92 and the library bound to neither STIV-infected cells nor normal cells. The initial library incubated with infected cells and aptamers incubated with noamal cells were used as the negative controls. c Fluorescent images of four FITC-aptamers with STIV-infected turtle tissues. QA-12 bound to STIV-infected liver and spleen tissues but not to normal tissues. QA-9, QA-36 and QA-92 bound to neither STIV-infected tissues nor normal tissues. The initial library was used as a negative control. Left: bright field; right: fluorescent. (Bars = 20 μm)
Mentions: To determine if aptamers against STIV recognized STIV-infected cells, we incubated FITC-aptamers (300 nM) with STIV-infected FHM cells and monitored the process by flow cytometry. The FITC-labeled initial library incubated with STIV-infected FHM cells were used as the control. Compared to the control, the fluorescence intensity of QA-12 increased in STIV-infected FHM cells, though the effects of QA-9, QA-36 and QA-92 were less noticeable, which means only QA-12 bound to STIV-infected cells, while QA-9, QA-36, QA-92 and the library could not bind to STIV-infected cells (Fig. 7a). These results were verified by imaging infected cell-aptamer interactions (Fig. 7b). Aptamer QA-12 evolved against STIV could thus recognize STIV-infected FHM cells. Fluorescent images of liver and spleen tissues indicated that the selected aptamers bound STIV-infected liver and spleen tissues with high levels of specificity (Fig. 7c).Fig. 7

Bottom Line: Soft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis).Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV.Aptamer QA-36 had the highest calculated binding affinity (K d ) of 53.8 nM.

View Article: PubMed Central - PubMed

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.

ABSTRACT

Background: Soft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis). More efficient methods of controlling and detecting STIV infections are urgently needed. 

Methods: In this study, we generated eight single-stranded DNA (ssDNA) aptamers against STIV using systematic evolution of ligands by exponential enrichment (SELEX).

Results: The aptamers formed representative stem-loop secondary structures. Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV. Aptamer QA-36 had the highest calculated binding affinity (K d ) of 53.8 nM. Flow cytometry and fluorescence microscopy of cell-aptamer interactions demonstrated that QA-12 was able to recognize both STIV-infected cells and tissues with a high level of specificity. Moreover, the selected aptamers inhibited STIV infection in vitro and in vivo, with aptamer QA-36 demonstrating the greatest protective effect against STIV and inhibiting STIV infection in a dose-dependent manner.

Discussion: We generated DNA aptamers that bound STIV with a high level of specificity, providing an alternative means for investigating STIV pathogenesis, drug development, and medical therapies for STIV infection.

Conclusions: These DNA aptamers may thus be suitable antiviral candidates for the control of STIV infections.

No MeSH data available.


Related in: MedlinePlus