Detection of Betanodavirus in experimentally infected European seabass (Dicentrarchus labrax, Linnaeus 1758) using non‐lethal sampling methods

Abstract

One of the major disease threats affecting the Mediterranean aquaculture industry is viral encephalopathy and retinopathy (VER). The target organs for Betanodavirus detection are the brain and eyes, obtained through lethal sampling. This study aimed to evaluate the efficacy and suitability of non‐lethal samples for detecting Betanodavirus in European seabass (Dicentrarchus labrax). European seabass juveniles were infected with Betanodavirus, by either an intramuscular injection or immersion (107 TCID50/ml and 106 TCID50/ml, respectively), and samples collected 7, 15 and 30 days post‐infection (dpi). The brain was collected as a lethal sample, and gills, caudal fin and blood as non‐lethal tissues for detecting Betanodavirus by quantitative reverse transcription PCR (RT‐qPCR). The presence of virus in non‐lethal tissues was inconsistent, with lower viral loads than in the brain. For blood, higher viral loads were detected in intramuscular‐infected fish at 15 dpi until the end of the challenge. Serum antibodies against Betanodavirus were assessed using an enzyme‐linked immunosorbent assay (ELISA). Antibodies were detected as early as 7 dpi, with higher mean antibody titres at 15 and 30 dpi. The presence of Betanodavirus‐specific antibodies indicates that this is a suitable evaluation method for detecting early stages of the infection.

Investigation of routes of entry and dispersal pattern of RGNNV in tissues of European sea bass, Dicentrarchus labrax

“…Investigation of viral genome in blood samples by molecular methods has been considered as a non‐lethal method for the detection of betanodaviruses despite the lower sensitivity compared to brain samples (Dalla Valle et al., 2000, 2005; Ferreira, Costa, Macchia, Dawn Thompson, & Baptista, 2019; Korsnes, Karlsbakk, Devold, Nerland, & Nylund, 2009; Olveira, Soares, Engrola, Dopazo, & Bandín, 2008; Tarrab, Ravid‐Peretz, & Ucko, 2019). According to our findings, the late development of viraemia accompanied by the significant lower RNA copies number obtained in the blood samples than those detected in the neuronal tissues indicate that the use of blood samples for the evaluation of the free status of fish during the early stages of RGNNV infection is not reliable.…”

Betanodavirus and VER Disease: A 30-year Research Review

“…In addition, a real time procedure combined with high resolution melting (HRM) has been proposed for NNV detection and genotyping [274]. RT-qPCR has also used for non-lethal detection, testing blood, gills and caudal fin samples [282].…”

Integrated Management Strategies for Viral Nervous Necrosis (VNN) Disease Control in Marine Fish Farming in the Mediterranean

“…However, in this case, it is important to stress that the predictive value of blood samples in the detection of the virus is lower due to problems related to the poor stability of the viral RNA in refrigerated samples, the need for specific extraction protocols and also because viruses are only present in the blood during viraemic stages. The presence of virus in non-lethal tissues including blood was inconsistent even after experimental infection [ 68 ]. Detection of antibodies against betanodavirus by ELISA (see the previous section on indirect diagnosis) and other methods were used in the past and still can be used to evaluate if the fish have been in contact with the virus and to complement other diagnostic techniques.…”