Antiviral activity is one of the most attractive applications of microalgal EPSs. Actually, an increasing number of studies proving the antiviral activity of these molecules is being published in last years (Shi et al., 2017; Pereira, 2018; Pereira and Critchley, 2020; Carbone et al., 2021). Most of these studies report positive results against a great number of important human viral pathogens, such as herpes simplex virus, human cytomegalovirus, influenza A virus, human immunodeficiency virus, human papillomavirus, dengue virus, rhinovirus, or even SARS-coV-2. In contrast, the EPS effect against fish viruses has been scarcely evaluated (Fabregas et al., 1999; Katharios et al., 2005; Jyotsna et al., 2021). In the present study, a clear antiviral effect of T. suecica and P. cruentum EPSs has been demonstrated against VHSV, an important pathogen for the aquaculture industry, supporting EPSs as major molecules in the biotechnological use of microalgae, and EPS antiviral activity as one of their most valuable applications in aquaculture.
Only one out of the eight different EPSs evaluated in this study was cytotoxic on RTG-2 cells: acid EPSs from heterotrophic cultures of P. cruentum (IC50 values < 100 µg mL-1). The lack of cytotoxicity of EPSs extracted from T. suecica and P. cruentum cultured in autotrophic and heterotrophic conditions has also been proven in mammalian cells; however, those substances are highly cytotoxic on tumoral mammalian cells, thus making these molecules attractive candidates for antitumor therapies (Parra-Riofrío et al., 2020; 2021). Interestingly, in all cases, important differences between acid and total EPS IC50 values were recorded (Table 2), showing acid EPSs from autotrophic cultures and total EPSs from heterotrophic cultures higher IC50 values. Differences in the effect of each EPS are probably due to their specific composition, which is quite different regarding content of proteins, carbohydrates, lipids and pigments, as well as monosaccharide composition (Parra-Riofrío et al., 2020; 2021).
The antiviral activity of the selected EPSs was evaluated by two parameters: viral genome replication, estimated by viral genome quantification in cells, and infective viral particle production, estimated by viral titres in supernatants. As expected, when viral genome replication was completely abolished, no infective viral particles were produced. This has been reported for T. suecica EPSs from autotrophic cultures in both, adsorption and post-adsorption assays (Figs. 1A, B; Figs. 2A, B), and for P. cruentum EPSs (heterotrophic and autotrophic) acting after viral adsorption (Fig. 4). When viral genome replication was only partially inhibited, as in the adsorption assay of T. suecica EPSs from heterotrophic cultures, a reduced production of infective viral particles was observed (Fig. 1C, D). Interestingly, even when viral genome replication was not affected (post-adsorption assay of T. suecica EPSs from heterotrophic cultures (Fig. 2C, D) the production of infective viral particles was reduced, which indicates that EPSs are interfering viral assembling and/or release. In the adsorption assays of P. cruentum EPSs (Fig. 3), genome amount was similar at 36 h p.i., but lower at earlier time points, thus suggesting a delay in viral genome replication that could be responsible for the lower production of infective viral particles (Fig. 3B, D).
Microalgal EPSs may exert their antiviral activity by interfering with different stages of the viral infection and replication process (Shi et al., 2017; Chen and Huang, 2018; Carbone et al., 2021). Regarding their anti-adsorption activity, EPSs can directly interact with viral particles, and/or can bind to cell receptors, competing with viral particles (Harden et al., 2009; Shi et al., 2017). The most evident effect on VHSV adsorption was recorded for T. suecica EPSs from autotrophic cultures (Fig. 1A, B). Furthermore, these EPSs also showed a strong post-adsorption effect (Fig. 2A, B). Although further experiments should be conducted to determine the mechanism or mechanisms involved in the observed effects, T. suecica EPSs from autotrophic cultures showed an attractive potential application to fight VHSV infections in aquaculture.
EPSs from heterotrophic cultures of T. suecica also showed adsorption and post-adsorption effects, although at a lower level than EPSs from autotrophic cultures (Figs. 1 and 2). In contrast, P. cruentum EPSs showed a much stronger post-adsorption effect, and no differences between EPSs from autotrophic or heterotrophic cultures were recorded (Figs. 3 and 4). These differences can be due to the specific composition of each EPS type. It has been proposed that post-adsorption antiviral effect of microalgal EPSs involves EPS interaction with cell surface receptors, which would activate the innate immune response, and/or would interfere viral genome uncoating, transcription, translation, and/or capsid assembly (Shi et al., 2017; Carbone et al., 2021).
The anti-VHSV activity of extracts from both species, T. suecica, and P. cruentum, has been previously tested (Fábregas et al., 1999). In contrast with our results, no antiviral activity was recorded for T. suecica extracts; however, P. cruentum extracts showed anti-VHSV adsorption activity. The cell line used (epithelioma papulosum cyprinid, EPC cells); the experimental design (adsorption assays, after overnight incubation of algal extracts with VHSV at 4 ºC); the parameter used to detect antiviral activity (number of infected cells foci detected by immunostaining); and, more importantly, the low purification level of microalga extracts, could be responsible for the different results recorded. In previous studies, P. cruentum extracts had shown activity against African swine fever virus, Herpes simplex viruses (type 1 and 2) and Varicella zoster virus (Fábregas et al., 1999; Huleihel et al., 2001). In contrast, to the best of our knowledge, this is the first report on T. suecica antiviral activity, although previous studies had demonstrated its activity against bacterial fish and crustacean pathogens (Austin and Day, 1990; Austin et al., 1992).
In summary, a clear antiviral activity of EPSs from T. suecica and P. cruentum against VHSV has been demonstrated. T. suecica EPSs from autotrophic cultures showed the strongest effect, as both, adsorption and post-adsorption phases of VHSV multiplication cycle in RTG-2 cells were affected. These results pave the way to use microalgal EPSs to fight viral diseases in aquaculture. To that end, it would be necessary to conduct studies focused on (i) the identification of EPS bioactive molecules; (ii) disclosing the underlying mechanisms of the antiviral activity; (iii) in vivo assays; and (iv) evaluation of EPS antiviral activity against other viral pathogens relevant for the aquaculture industry.