Nguyen, Ngoc-Tran T., Minami, Junya, Andrade Martinez, Maria, Knuckey, Richard M., Zenger, Kyall R., Jerry, Dean R., and Condon, Kelly (2026) 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. Aquaculture, 623. 744129.

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Abstract

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 × 10⁷, 4.6 × 10⁶, 4.6 × 10⁵ and 4.6 × 10⁴ 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 × 10⁴ to 100% at 4.6 × 10⁷ 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 10¹² RGNNV copies/g, whereas subclinically affected injected fish displayed lower RGNNV detection frequency and copy number (< 10⁷ 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.

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