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<p>African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating disease of domestic pigs with mortality rates approaching 100%, leading to severe global economic losses. No effective vaccines or antivirals are available, highlighting the urgent need for novel therapeutic strategies and efficient screening tools. We developed a recombinant ASFV-expressing dual reporter (<i>mCherry</i> and <i>NanoLuc</i>), rASFV_mChNluc, and established a high-content screening (HCS) platform optimized for cost-effective, low-labor analysis via the mCherry reporter. The assay demonstrated excellent robustness (Z′-factor = 0.669±0.064) and successfully verified the activity of the known ASFV inhibitor AraC, confirming inhibition at the postinfection stage. Screening of an in-house fungal extract library (493 extracts) identified 25 hits (5.07%) that reduced viral infectivity to &lt; 5%. Extracts from the insect fungi <i>Beauveria neobassiana</i> and <i>Samsoniella aurantia</i> showed potent activity, with an SI &gt; 62.81 (EC₅₀ = 1.99±0.71&#xa0;µg/mL) and an SI &gt; 42.92 (EC₅₀ = 11.65±2.99&#xa0;µg/mL), respectively. Time-of-addition assays indicated that <i>B. neobassiana</i> acts at multiple replication stages, whereas <i>S. aurantia</i> targets the postinfection stage. This study establishes a robust ASFV HCS platform for efficient high-throughput antiviral discovery and highlights fungi as a promising source of novel ASFV inhibitors.</p>

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A high-content screening platform for antiviral drug discovery using a recombinant African swine fever virus

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月7日

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  • Challika Kaewborisuth,
  • Artit Khonsanit,
  • Donnaya Thanakitpipattana,
  • Nathiphat Tanwattana,
  • Benjamas Liwnaree,
  • Jennifer Luangsa-ard,
  • Nopporn Chutiwitoonchai

摘要

African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating disease of domestic pigs with mortality rates approaching 100%, leading to severe global economic losses. No effective vaccines or antivirals are available, highlighting the urgent need for novel therapeutic strategies and efficient screening tools. We developed a recombinant ASFV-expressing dual reporter (mCherry and NanoLuc), rASFV_mChNluc, and established a high-content screening (HCS) platform optimized for cost-effective, low-labor analysis via the mCherry reporter. The assay demonstrated excellent robustness (Z′-factor = 0.669±0.064) and successfully verified the activity of the known ASFV inhibitor AraC, confirming inhibition at the postinfection stage. Screening of an in-house fungal extract library (493 extracts) identified 25 hits (5.07%) that reduced viral infectivity to < 5%. Extracts from the insect fungi Beauveria neobassiana and Samsoniella aurantia showed potent activity, with an SI > 62.81 (EC₅₀ = 1.99±0.71 µg/mL) and an SI > 42.92 (EC₅₀ = 11.65±2.99 µg/mL), respectively. Time-of-addition assays indicated that B. neobassiana acts at multiple replication stages, whereas S. aurantia targets the postinfection stage. This study establishes a robust ASFV HCS platform for efficient high-throughput antiviral discovery and highlights fungi as a promising source of novel ASFV inhibitors.