Generation and identification of novel DNA aptamers with antiviral activities against largemouth bass virus (LMBV)

Abstract

Largemouth bass virus (LMBV) causes high mortality in largemouth bass, and results in serious economic loses in the aquaculture industry. However, there is no effective treatment to control LMBV infection. Aptamers are single-stranded oligonucleotides with high specificity and affinity to various targets, including viruses, viral components, and virus-infected cells. In this study, three novel DNA aptamers (LA38, LB49, and LA13), which specifically targeted LMBV, were identified by selective evolution of ligands by exponential enrichment. These aptamers formed stable stem-loop structures identified by secondary structures and minimum free energy predictions. Moreover, enzyme-linked apta-adsorption assays, dissociation constant (Kd) measurements, and flow cytometry analyses showed that these aptamers specifically bound to LMBV with high affinity at the nanomolar level. Furthermore, these aptamers were nontoxic, and significantly inhibited LMBV infection in vitro and in vivo. The mortality of largemouth bass was reduced approximately 40% after 10 days of infection by LMBV preincubated with LA38 or LB49, compared to that infected with wild or random ssDNA pretreated LMBV. In addition, confocal imaging and flow cytometry analyses indicated that these aptamers only entered cells along with LMBV, suggesting their potential as a delivery system. Presently, this is the first report of specific aptamers targeting iridovirus from freshwater fish. These aptamers have great potential in diagnosis and therapy, and in delivery agents for detecting LMBV infection.