In-vivo evaluation of silver nanoparticles-enhanced Chlorella protein nanovaccine for immunological defense in grouper (Epinephelus sp) against RNA viral infections.

Viral RNA diseases, notably Viral Nervous Necrosis (VNN), routinely produce very high losses (≈80-100% mortality) in cultured groupers (Epinephelus sp.), driving major economic impacts across Asia. Conventional vaccination has been unreliable, largely because of antigen instability and suboptimal, short-lived immunity. Nanovaccine platforms-here, Chlorella-derived proteins augmented with silver nanoparticles (AgNPs)-offer a route to better antigen delivery and immune modulation in aquatic hosts. This study evaluated the efficacy of an AgNPs- Chlorella vulgaris protein nanovaccine in VNN-challenged groupers to enhance growth, hematology, survival, and tissue integrity. Eight cohorts were examined: two controls (K-, K+) and six dosing regimens (T1-T6). Fish received the nanovaccine by injection, followed by viral challenge. Key endpoints included hematological indices (RBC, WBC, hemoglobin, hematocrit, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), histopathology of intestine and kidney, and flow-cytometric profiling of CD4, CD8, IgM, growth (length, weight), survival rate (SR), and relative percent survival (RPS). Nanovaccination significantly enhanced performance and immunity. The T3 dose yielded the best outcomes, with body length 12.73 ± 0.87 cm and weight 18.00 ± 1.50 g, SR = 90% and RPS = 82%. Hematology improved (RBC 2.52 × 106 cells/μL, WBC 1.76 × 103 cells/μL, hemoglobin 7.4 g/dL, MCV 102 μm3, MCHC 26.4%). Immune readouts showed CD4+ 13.44%, CD8+ 8.48%, and IgM 4.25%. Fish tissue showed milder lesions (≤25% damage) compared to those infected with severe injury virus (>45%). The Nanovaccine supports its potential as environmentally friendly and long-lasting means of protecting marine aquaculture from disease. This work supports the objectives of SDG 14, emphasizing the conservation of life below water.