A cell-penetrating peptide Pep-1 fused nanobody Nb24 targeting VP28 inhibits WSSV replication in crayfish Procambarus clarkii

作者：Wei X, Wei S, Yi C, Luo S, Wei T, Huang J, Xie L, Ma Z, He W, Wang Y, Huang A · 发表于：Fish & shellfish immunology · 年份：2026 · DOI：10.1016/j.fsi.2026.111575 · 研究领域：Astacoidea、White spot syndrome virus 1、Virus Replication、Single-Domain Antibodies、Viral Envelope Proteins、Peptides、Cell-Penetrating Peptides、Antiviral Agents、Animals、Arthropod Proteins、Cysteamine

White spot syndrome virus (WSSV) is a fatal pathogen responsible for significant economic losses in the global crustacean industry, and there are no effective commercial therapeutic drugs available for controlling the spread of WSSV. Nanobodies (Nbs) have attractive antiviral properties including small molecular size, high specificity, stable structure, and ease of production. Cell-penetrating peptide Pep-1 can serve as a vector to deliver proteins into cells. In our previous study, a nanobody named Nb24 targeting WSSV envelope protein VP28 was successfully screened using phage display technology. In this study, Nb24 was fused to Pep-1 to generate Pep-1-Nb24, and the entry efficiency of Pep-1-Nb24 and its anti-WSSV activity were evaluated in crayfish Procambarus clarkii. The results showed that Nb24 could significantly block WSSV infection in a dose-dependent manner in crayfish, and improve the survival rate of WSSV-infected crayfish. Nevertheless, the poor membrane permeability of Nb24 made it difficult to enter cells autonomously. Interestingly, Pep-1 successfully delivered Nb24 into hemocytes and tissues of the gill and hepatopancreas, and Pep-1-Nb24 had a stronger anti-WSSV activity in crayfish. Moreover, the expression of antioxidant genes SOD, CAT, GST and immune genes Lzm, Crustin, and Propo was significantly upregulation after treatment with Nb24 and Pep-1-Nb24 in WSSV-infected crayfish. The expression levels of these genes in Pep-1-Nb24 groups were higher than those ...