Introduction
Viral haemorrhagic septicaemia virus (VHSV) infects a wide range of freshwater and marine fish species, causing high mortality rates in susceptible cultured fish, mainly salmonids (Smail and Snow, 2011). A prophylactic practise to enhance the natural resistance of farmed fish to viral infections is the use of functional food. Extracellular polymers (ECPs) from probiotic bacteria are postbiotics that represent a valid and safe alternative for providing beneficial effects against microorganisms in fish (Domínguez-Maqueda et al., 2024; García-Márquez et al., 2023). Macroalgae and microalgae are also alternative sources of substances with potential activity to improve resistance to microbial infections (Abdala-Díaz et al., 2021; Parra-Riofrio et al., 2023). The objective of this study was to evaluate the antiviral activity of ECPs from the probiotic bacteria Shewanella putrefaciens Pdp11 and Bacillus pumilus UMA169, as well as extracts and/or polysaccharides from Ulva rigida and Porphyridium cruentum, against a freshwater VHSV isolate.
Materials and methods
The protection conferred by each substance against a freshwater VHSV isolate (genotype 1) was evaluated in vitro, on RTG-2 cells, by quantifying the cellular survival rate after viral infection, using the MTT reduction assay. Cells were inoculated at 0.05 MOI, and, after an adsorption time of 1 hour, the viral suspension was removed, and replaced by cultured medium supplemented with the substances. Cells were cultured at the optimum temperature for viral replication, and the cellular survival rate was calculated 3 days post-infection. The substances tested were: (i) ECPs from B. pumilus UMA169, ECPs from S. putrefaciens Pdp11 (both bacteria cultured on TSAs at 15 ºC for 48 h) at 50 μg of protein·mL-1, (ii) extracts of U. rigida at 100 and 500 μg·mL-1, (iii) ulvan polysaccharides from U. rigida at 50 μg·mL-1 and (iii) extracts of P. cruentum at 100 μg·mL-1.
Results
All substances tested exhibited antiviral activity against VHSV, except for ECPs from Pdp11, which showed the opposite effect (Figure 1). ECPs from B. pumilus UMA169 conferred full protection to the cells, whereas algal extracts and ulvan polysaccharides conferred a similar protection level to VHSV infected cells. Thus, these substances, excluding ECPs from Pdp11, are promising candidates to be used as protective substances against VHSV. Actually, their effect on viral replication and infective viral particles production will also be evaluated.
According to these results, a supplemented diet will be designed to test the anti-VHSV activity of these substances in the experimental model of zebrafish to evaluate their antiviral potential and relevance to aquaculture.
References
- Abdala-Díaz, et al. (2021). Effects of a short pulse administration of Ulva rigida on innate immune response and intestinal microbiota in Sparus aurata juveniles. Aquaculture Research, 52(7), 3038–3051.
-Domínguez-Maqueda, et al. (2024). Evaluation of the Differential Postbiotic Potential of Shewanella putrefaciens Pdp11 Cultured in Several Growing Conditions. Marine Biotechnology (New York, N.Y.), 26(1), 1–18.
-García-Márquez, et al. (2023). Potential Effects of Microalgae-Supplemented Diets on the Growth, Blood Parameters, and the Activity of the Intestinal Microbiota in Sparus aurata and Mugil cephalus. Fishes, 8(8), 409.
-Parra-Riofrio, et al. (2023). Tetraselmis suecica and Porphyridium cruentum exopolysaccharides show anti-VHSV activity on RTG-2 cells. Aquaculture International, 31(6), 3145–3157.
-Smail and Snow, (2011). Viral haemorrhagic septicaemia. In P. T. K. Woo & D. W. Bruno (Eds.), Fish diseases and disorders. Volume 3: viral, bacterial and fungal infections. (2nd ed., Vol. 3, pp. 110–142). CABI International.
Acknowledgments
Project PCM_000035, funded with resources from the "Next Generation EU" Recovery Fund through the Recovery, Transformation and Resilience Plan” and co-funded by “Consejería de Universidad, Investigación e Innovación”, from the Regional Government of Andalusia. The opinions expressed are solely those of the author(s) and do not necessarily reflect those of the European Union or the European Commission. The European Union and the European Commission are not responsible of these opinions.