Red-spotted Grouper Nervous Necrosis Virus (RGNNV) detection in giant grouper (Epinephelus lanceolatus) using destructive and non-destructive samples is influenced by infectious dose, challenge route, and infection outcome

Viral Encephalopathy and Retinopathy (VER), caused by Red-spotted Grouper Nervous Necrosis Virus (RGNNV), is a major constraint on giant grouper ( Epinephelus lanceolatus ) aquaculture. Current surveillance depends on destructive brain and eye sampling, limiting scalable and repeated monitoring of broodstock and high-value fish. This study evaluated detection performance of destructive and non-destructive sampling methods for RGNNV in giant grouper following experimental intramuscular injection challenge. One hundred juvenile giant groupers were challenged with four RGNNV doses (4.6 × 10 7 , 4.6 × 10 6 , 4.6 × 10 5 and 4.6 × 10 4 copies/mL) in duplicate tanks, each containing five cages (10 fish/cage) representing four dose groups and one cohabitated control. RGNNV copy number was quantified by RT-qPCR across nine destructive tissues (brain, eyes, nostril, gill, head kidney, spleen, injection-site muscle and gut), two non-destructive samples (mucous and urine), and parallel tank water environmental RNA (eRNA). Fish morbidity increased dose-dependently, from 30% at 4.6 × 10 4 to 100% at 4.6 × 10 7 copies/mL dose. RGNNV detection and copy number also aligned with clinical outcome. Clinically affected injected fish exhibited a clear neural tropism, with brain and eyes reaching up to 10 12 RGNNV copies/g, whereas subclinically affected injected fish displayed lower RGNNV detection frequency and copy number (< 10 7 copies/g). Horizonal transmission was confirmed, with 5% morbidity in cohabitated fish. Among non-destructive samples, only mucous consistently detected RGNNV (100% in clinically affected injected fish and 69.2% in subclinically affected injected fish), while urine had no detection, confirming poor diagnostic value. eRNA detected RGNNV from day 7 post-challenge, peaking after fish morbidity and reflecting active shedding rather than early detection. These findings highlighting the diagnostic value of mucous sampling and the complementary role of eRNA in surveillance of giant grouper aquaculture. • Destructive and non-destructive samples were evaluated for RGNNV detection in giant grouper. • Brain and eye exhibited the highest RGNNV copy number in affected fish. • Mucous reliably detected RGNNV in clinical and subclinical fish. • Urine displayed no diagnostic value for RGNNV detection. • eRNA monitoring reflected tank-level RGNNV shedding during infection.