Peer Review #1 of "Evaluation of a new Argovit as an antiviral agent included in feed to protect the shrimp Litopenaeus vannamei against White Spot Syndrome Virus infection (v0.1)"

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 antiviral 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 shrimp infectious doses (SID) were exposed to 40, 100, 200 and 1000 ng/SID of Ag+ and then intramuscularly injected into shrimp for 96 h.In assay 2, shrimp were fed Argovit-4 supplemented in feed at different concentrations (10, 100 and 1000 µg per gram of feed) for 192 h.In assay 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 the 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