In this study, four experimental assays were conducted to evaluate the use of a new silver nanoparticle formulation named Argovit-4, which was prepared with slight modifications to enhance its biological activity against white spot syndrome virus (WSSV) in shrimp culture. The goals of these assays were to (1) determine the protective effect of Argovit-4 against WSSV, (2) determine whether Argovit-4 supplemented in feed exhibits toxicity towards shrimp, (3) determine whether Argovit-4 as

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... l additive in feed can prevent or delay/reduce WSSV-induced shrimp mortality, and (4) determine whether Argovit-4 supplemented in feed alters the early stages of the shrimp immune response. In bioassay 1, several viral inocula calibrated at 7 SID 50 (shrimp infectious doses 50% endpoint) were exposed to 40, 100, 200 and 1,000 ng/SID 50 of Ag + and then intramuscularly injected into shrimp for 96 h. In bioassay 2, shrimp were fed Argovit-4 supplemented in feed at different concentrations (10, 100 and 1,000 µg per gram of feed) for 192 h. In bioassay 3, shrimp were treated with Argovit-4 supplemented in feed at different concentrations and then challenged against WSSV for 192 h. In bioassay 4, quantitative real-time RT-qPCR was performed to measure the transcriptional responses of five immune-relevant genes in haemocytes of experimental shrimp treated with Argovit-4 supplemented in feed at 0, 6, 12, 24 and 48 h. The intramuscularly injected Argovit-4 showed a dose-dependent effect (p < 0.05) on the cumulative shrimp mortality from 0-96 h post-infection. In the second bioassay, shrimp fed Argovit-4 supplemented in feed did not show signs of toxicity for the assayed doses over the 192-h experiment. The third and fourth bioassays showed that shrimp challenged with WSSV at 1,000 µg/g feed exhibited reduced mortality without altering the expression of some immune system-related genes according to the observed level of transcriptional. This study is the first show that the new Argovit-4 formulation has How to cite this article Romo-Quiñonez CR, Álvarez-Sánchez AR, Álvarez-Ruiz P, Chávez-Sánchez MC, Bogdanchikova N, Pestryakov A, Mejia-Ruiz CH. 2020. Evaluation of a new Argovit as an antiviral agent included in feed to protect the shrimp Litopenaeus vannamei against White Spot Syndrome Virus infection. PeerJ 8:e8446 http://doi.org/10.7717/peerj.8446 potential as an antiviral additive in feed against WSSV and demonstrates a practical therapeutic strategy to control WSSV and possibly other invertebrate pathogens in shrimp aquaculture. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.8446#supplemental-information. REFERENCES Abad-Rosales SM, Frías-Espericueta MG, Romero-Bernal OG, Lozano-Olvera R, Garcia-Gasca SA, Montoya-Rodriguez L, Voltolina D. 2019. White spot syndrome virus (WSSV) infection and immunity responses in white shrimp (Litopenaeus vannamei) exposed to sublethal levels of metals. Aquaculture Research 50:758-764 R. 2017. Antibacterial effect of biosynthesized silver nanoparticles in Pacific white shrimp Litopenaeus vannamei (Boone) infected with necrotizing hepatopancreatitis bacterium (NHP-B). Latin American Journal of Aquatic Research 45(2):421-430 DOI 10.3856/vol45-issue2-fulltext-17. Akter N, Alam J, Jewel AS, Khatun S, Akter S. 2018. Evaluation of dietary metallic iron nanoparticles as feed additive for growth and physiology of Bagridae catfish Clarias batrachus (Linnaeus, 1758). International Journal of Fisheries and Aquatic Studies 6(3):371-377. Álvarez Ruiz P, Mejía-Ruiz CH, Magallón-Barajas FJ, Escobedo-Bonilla CM. 2013. Silencing Pacific white shrimp, Litopenaeus vannamei LvRab7 reduces mortality in brooders challenged with white spot syndrome virus. Aquaculture Research 44:772-782