The Nodaviridae family has been causing major damage affecting species such as grouper, Asian seabass/barramundi, European sea bass, gilthead sea bream, Japanese and barfin flounder, Atlantic and Pacific cod and Atlantic halibut (Bandín and Souto, 2020). The knowledge on antiviral activity of probiotics has been raised in recent years (Lakshmi et al., 2013). For example, Pseudomonas, Vibrio, Aeromonas spp. and Coryneforms had antiviral activity against infectious hematopoietic necrosis virus (IHNV) (Kamei et al., 1988). Previously, Li et al. (2009) demonstrated that feeding with a Bacillus megaterium strain increased the resistance to white spot syndrome virus (WSSV) in the shrimp Litopenaeus vannamei. Application of Latic Bacteria probiotics (LAB) as a single strain or mixed with Sporlac improved disease resistance against lymphocystis viral disease in olive flounder (Paralichthys olivaceus) (Harikrishnan et al., 2010). Among invertebrates, probiotic bacteria have been found to induce both cellular and humoral immune responses in the shrimp (Castex et al., 2008, Farzanfar, 2006, Vargas-Albores et al., 2017, Knipe et al., 2021). This includes the stimulation of pro-inflammatory cytokines, increasing the phagocytic activity of leucocytes (Pirarat et al., 2006), increasing the levels of antibodies complements (Balcázar et al., 2007), cytokines (interleukin-1 (IL-1), IL-6, IL-12, tumor necrosis factor a (TNF-a), gamma interferon (IFN-g), IL-10 and antimicrobial peptides (Mukherjee et al., 2012). In the current study, assays were implemented in order to further refine work initiated to demonstrate the potential use of novel lactic acid bacteria isolated from mucus of freshwater fish (Mugil cephalis) and involved as probiotic in aquaculture (El-Jeni et al., 2019) seeking to address more gaps such as exploring the anti- nodavirus RGNNV activity. By primary analyses, the first experiment did not detect any toxicity following R.A73 E. Faecium application on E-11 fish cell line and no morphological differences were observed, giving evidence of cell biosafety. This has been seen in the case of Chai et al. (2013), at concentrations up to 1.00E + 07 CFU/ml of E. faecium NCIMB 10415 in animal nutrition. In addition, the release of TNF-α, cytokine IL-10 and CCR3 genes were monitored to determine the underlying cellular mechanism. The E-11 cells were treated with R.A73 E. faecium resulting an over-expression of the IL-10 gene. We can assume that the response of the innate immune system, characterized by the increase of the anti-inflammatory cytokine IL-10 caused the beneficial physiological effects of the probiotic Enterococcus strain. This result is in agreement with Tarasova et al. (2010) findings which showed the influence of probiotic E. faecium strain on cytokines expression in rats and as an optimal supplementation level of dietary for tilapia (Li et al., 2020). In contrast to earlier findings, however, no evidence of TNF-α secretion was detected in the present study, the ratios obtained from control cells compared to R.A73 E. faecium-inoculated wells revealed a decline in TNF-α expression levels. These results reflect those of Kern et al. (2017) who also found that upregulation of TNF-α at the mRNA level was prevented when cells were co-incubated with E. faecium. On the other hand, the assessment of gene expression coding for the chemokine receptor C-C chemokine receptor 3 (CCR3) from cells inoculated with R.A73 E. faecium showed increased expression of the CCR3. In previous study, the probiotic treatment altered markers such as CCR3, resulting in an overall increase in gene expression, which was particularly noticeable in fish fed diets (Moroni et al., 2021).
When investigating the antiviral activity, the R.A73 E. faecium was added for 1h right before the infection period by the nodavirus. The findings revealed a substantial decrease in the expression of IL-10, TNF-α, and the CCR3 genes compared to E-11 cells infected with nodavirus alone. We hypothesis the protective effect of the probiotic R.A73 E. Faecium on cells. Indeed, IL-10, a prototypical anti-inflammatory cytokine, produced by activated monocytes, T cells and other cell types such as keratinocytes, is considered to be a crucial factor in a major immune response and inflammation inhibitor (Brockmann et al., 2018). We speculated that the probiotic R.A73 E. faecium has a protective effect on the cells and reduces the inflammatory cytokine expression induced by nodavirus infection. Hence, by co-incubating with this probiotic strain, the stimulus for the release of IL-10 is blocked and induced an under expression of TNF-α and CCR3 compared to the virus-infected cells during the seven days of post-inoculation assuming the probiotic to have protective effects on cells.
Altogether, further studies are needed to support data obtained. The first evidence-based capability of R.A73 E. faecium novel probiotic strain to induce INF-a, IL-10 and CCR3 immune genes in E-11 cells may subsequently contributed to improving the antiviral response mounted by the cells against RGNNV nodavirus.