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IntroductionDue to the impact of antibody-dependent enhancement and viral variation, effective vaccines or antiviral therapies remain lacking for the dengue virus (DENV). Nucleic acid drugs, particularly Vivo-Morpholinos (MOs), have emerged as a promising avenue for antiviral treatment due to their programmability and precise targeting, as well as their safety and stability.MethodIn this study, we designed and developed 10 morpho-modified (octa-guanidine dendrimer) vivo-MO molecules that target each coding gene of DENV. Subsequently, we assessed the inhibitory impact of vivo-MOs on dengue viral RNA load utilizing qRT-PCR. Furthermore, we examined the inhibitory effect on the live virus through a plaque assay and the TCID50 assay.ResultsWe found that the vivo-3′UTR molecule targeting the 3′ untranslated region of the dengue virus exhibited the highest inhibitory rate against viral load. The vivo-3′UTR demonstrated 99% inhibition of dengue virus RNA and the inhibition of up to 98% of the live virus. Additionally, the targeted sequence was conserved among all four DENV serotypes, and treatment with 10 μM of vivo-3′UTR resulted in a reduction of viral titers for all four DENV serotypes by over 99.99%. Additionally, we revealed that pre-treatment with vivo-3′UTR had a notable preventive effect against viral infection.ConclusionThis study screened an effective vivo-MO target drug for the treatment of dengue virus infection, demonstrating low toxicity in mammalian cell lines, and proposed a novel preventive antiviral approach.

IntroductionRapid and high-throughput screening of antiviral clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (crRNAs) is urgently required for the CRISPR-Cas13a antiviral system. Based on the same principle, we established an efficient screening platform for antiviral crRNA through CRISPR-Cas13a nucleic acid detection.MethodIn this study, crRNAs targeting PA, PB1, NP, and PB2 of the influenza A virus (H1N1) were screened using CRISPR-Cas13a nucleic acid detection, and their antiviral effects were confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RNA secondary structures were predicted by bioinformatics methods.ResultsThe results showed that crRNAs screened by CRISPR-Cas13a nucleic acid detection could effectively inhibit viral RNA in mammalian cells. Besides, we found that this platform for antiviral crRNA screening was more accurate than RNA secondary structure prediction. In addition, we validated the feasibility of the platform by screening crRNAs targeting NS of the influenza A virus (H1N1).DiscussionThis study provides a new approach for screening antiviral crRNAs and contributes to the rapid advancement of the CRISPR-Cas13a antiviral system.