Development of a High-Throughput Screening Method for Anti-NNV Drugs

Nervous necrosis virus (NNV) is an infectious pathogen, characterized by rapid infection and high mortality, commonly encountered in the aquaculture industry. Therefore, there is an urgent need to develop effective antiviral drugs against NNV, which is crucial for targeted treatment strategies, pathogen control, and loss reduction. Screening of effective antiviral active compounds, namely drug precursors, is key to developing highly efficient drugs against NNV. Reverse transcription-quantitative real-time PCR (RT-qPCR) is widely applied to screen and evaluate various substances to prevent and control virus infection. However, RT-qPCR is a cumbersome procedure and not suitable for high-throughput rapid screening. The aptamer TNA1c, which specifically binds to NNV-infected cells, was used to construct a target-driven activatable aptamer probe (TAA). Then, the TAA probe was applied to establish a high-throughput screening (TAA-HTS) method for efficient evaluation of substances against NNV infection. TAA-HTS technology achieved rapid, sensitive, and specific screening of bioactive substances with significant anti-NNV effects. As compared to commonly used analytical methods, such as RT-qPCR, TAA-HTS has the advantages of easy operation and high sensitivity and specificity. The findings of this study provide data support and a theoretical basis for the development of effective antivirus preparations. IMPORTANCEIn this study, a target-driven activatable aptamer probe was employed to establish a high-throughput screening method for anti-NNV drugs. This method enables rapid, sensitive, and specific screening of anti-NNV compounds while reducing background interference. Although RT-qPCR is currently the most widely used technique for antiviral drug screening and validation, the screening approach developed in this study significantly shortens screening time while achieving results consistent with those of RT-qPCR. This demonstrates that the screening method presented here has the potential to become a universal strategy for future drug screening against viral diseases. Moreover, this study not only provides a novel technical solution for NNV control but also demonstrates the potential of aptamer-based probes in high-throughput antiviral screening, serving as a model for combating viral pathogens in aquaculture and other fields.