Newcastle disease (ND) is one of the most serious infectious diseases affecting birds, particularly poultry, and it has been the cause of serious economic losses . There is no treatment for ND. Vaccination is practiced widely, and it remains the recommended method for prevention. Although vaccination using live and killed vaccines is widely used as a management practice, the velogenic strains are endemic in the commercial poultry of many countries [15, 16]. This represents a major problem for the poultry industry and therefore requires further measures to prevent and control this disease.
Antiviral treatments are not available in poultry due to their cost and toxicity, and thus, a promising alternative is the use of compounds of natural origin, which have been underexplored for this purpose [17, 18]. Several sulfated seaweed polysaccharides have been demonstrated to exhibit antiviral activity against a wide spectrum of viruses [4, 5, 7, 19] with effectiveness that depends on the sugar composition, main chain length, sulfation level, and sulfate pattern [20, 21]. An advantage of sulfated polysaccharides from algae is the high content of polyanions in their extracellular matrix; consequently, they can be prepared and made available at a very low cost, and sulfated polysaccharides isolated from many marine algae possess species-specific structural variations that appear to affect their antiviral activity .
The major undesirable side effect of sulfated polysaccharides is their well-known anticoagulant activity. This adverse effect can be avoided by selecting sulfated polymers, such as fucoidan from C. okamuranus, which exhibit virtually no anticoagulant activity . Cumashi et al. suggested that the high presence of glucuronic acid branches is the most likely feature responsible for the lack of anticoagulant activity by C. okamuranus fucoidan, as the less active compounds are characterized by a low degree of sulfation and a high presence of 2-O-α-D-glucuronyl substituents along the linear polysaccharide backbone (Characteristics fucoidan from C. okamuranus) . By contrast, it has been proposed that the antiviral activity of fucoidan is related to the concentration of fucose and uronic acids. Hidari et al. found that the antiviral properties of fucoidan from C. okamuranus against dengue virus type 2 vanished when the glucuronic acid was carboxyl-reduced , and Jiao et al. indicated that a carboxyl-reduced fucoidan derivative from C. okamuranus, which contained fucose and sulfate groups but no uronic acids, did not display significant antiviral effects against influenza A . In the present study, we evaluated the antiviral activity of fucoidan from C. okamuranus extracted by the method of Tako et al. with a significant degree of uronic acids, and investigated its possible mechanism of action in Vero cells .
There are very few drugs available for the treatment of infections caused by RNA viruses. Ribavirin inhibits many paramyxoviruses in vitro, such as parainfluenza, the measles virus, the mumps virus, RSV, and canine distemper virus [24, 25]. Ribavirin is approved for the treatment of RSV infection in children; however, the efficacy of ribavirin against RSV is limited . In our work, ribavirin did not display good antiviral activity because its IC50 was 490, and its CC50 was close to that value (SI50=0.78). Conversely, according the findings, no significant in vitro toxicity was observed with fucoidan, as its CC50 was >1500 μg/mL. Previous studies reported low in vitro cytotoxicity for sulfated polysaccharides from different algal sources, consistent with our findings for fucoidan . Additionally, in the study by Gideon and Rengasamy, rats treated with C. okamuranus exhibited no necropsy or other pathological changes in organs or changes in histopathological morphology, consistent with the lower toxicity possessed by sulfated polysaccharides, specifically fucoidan from C. okamuranus.
Fucoidan from C. Okamuranus exhibited good antiviral activity against NDV, with an SI50 of >25.8, although the compound displayed a much greater ability to inhibit syncytia formation, with a calculated SI50 of >2000. Therefore, fucoidan from C. okamuranus much more effectively inhibited NDV. Similar results were obtained for other sulfated polysaccharides from different algae against several enveloped viruses [5, 19, 26, 29]; however; it appears clear that specific interactions between viruses and sulfated polysaccharides are related to the particular characteristics of the viruses and compounds. Variations in the amino acid sequence of the viral envelope glycoproteins result in differential susceptibilities to the compounds that interact with them [30–33]. Studies of structures related with the antiviral effect of Cladosiphon fucoidan against dengue viruses strongly suggested that both glucuronic acid and sulfated fucose residues in fucoidan appear to critically affect its antiviral effect . Fucoidan from C. okamuranus exhibited significant antiviral activity against NDV infection in this study, therefore suggesting a specific inhibition of NDV infection in host cells.
We found a virucidal effect negligible of fucoidan against NDV virions, indicating that the inhibitory effects detected by the inhibition syncytia or plaque reduction assays were actually due to interference with some step of the NDV replication cycle. The lack of virucidal activity for fucoidan from C. okamuranus is in accordance with previous studies that found most algal sulfated polysaccharides cannot induce significant virion inactivation [34–36].
According our results obtained by time of addition assays, fucoidan exerted an inhibitory effect on the early phases of the virus-cell interaction (Figure 3). These results agree with previously reported data indicating that algae-derived sulfated polysaccharides can inhibit viral infection by interfering with the binding and penetration of the virus into cells [5, 37]. We further confirmed the antiviral activity by Western blotting using NDV-infected Vero cells that were treated with fucoidan at different times after infection. Our results clearly reveal marked inhibition of HN protein expression, with the greatest inhibition obtained at 15 min post-infection (Figure 5). These results support the ability of fucoidan to block the early stages of infection.
As judged by our findings, fucoidan acts in early steps of infection and inhibits syncytia formation. Therefore, we decided to determine the specific viral step(s) inhibited by fucoidan. The NDV envelope contains two proteins related to entry: the attachment protein HN and the fusion protein F. Fusion of the viral envelope with the cell membrane occurs after attachment via conformational changes in the F protein that are specifically arrested at 4°C. The addition of fucoidan after prebinding of NDV to cells effectively blocked viral infection (Figure 4). These findings suggest that fucoidan principally inhibits NDV infectivity by blocking one or more postbinding entry steps.
As described in this study, although the primary antiviral targets of fucoidan were suggested to be early stages of NDV infection, fucoidan inhibited the cell-to-cell spread of NDV when added to the medium at 12 h postinfection. Regarding avirulent NDV strains, fusion did not occur without the addition of trypsin in the fusion inhibition assays with NDV La Sota, and we only observed antiviral activity when fucoidan was added before F protein cleavage, indicating that the compound inhibits fusion, perhaps via a direct effect on the F0 protein (Figure 6). As a consequence of this mode of action, fucoidan both inhibits the penetration of NDV to cells and strongly suppresses NDV-induced syncytia formation between NDV-infected cells and uninfected cells, a process that drastically enhances NDV spread and infectivity. Our results agree with those of Parskaleva et al., who demonstrated the ability of an extract from Sargassum fusiforme to inhibit the entry events of HIV and the mechanism by which HIV infection spreads .
In the penetration inhibition assays, we observed a reduction in viral infection when fucoidan was added after adsorption. As the cellular receptor to which the virus binds is already known (Figure 4), this finding suggests that the inhibitory effect of fucoidan on the viral F protein may be principally responsible for this inhibition and not an effect on the cellular receptor (when the F protein is possibly exposed to the compounds). However, we cannot rule out an effect on the HN protein, as the fusion mediated by the NDV F protein absolutely requires the participation of the HN protein . Our findings suggest that the inhibition of virus fusion events (virus-cell and cell-cell) may be principally responsible for the inhibition of NDV infectivity by fucoidan from C. okamuranus.