Sulphated polysaccharides from Ulva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion, in NDV infection

Abstract

Sulphated polysaccharides (SP) extracted from seaweeds have antiviral properties and are much less cytotoxic than conventional drugs, but little is known about their mode of action. Combination antiviral chemotherapy may offer advantages over single agent therapy, increasing efficiency, potency and delaying the emergence of resistant virus. The paramyxoviridae family includes pathogens causing morbidity and mortality worldwide in humans and animals, such as the Newcastle Disease Virus (NDV) in poultry. This study aims at determining the antiviral activity and mechanism of action in vitro of an ulvan (SP from the green seaweed Ulva clathrata), and of its mixture with a fucoidan (SP from Cladosiphon okamuranus), against La Sota NDV strain. The ulvan antiviral activity was tested using syncytia formation, exhibiting an IC50 of 0.1 μg/mL; ulvan had a better anti cell-cell spread effect than that previously shown for fucoidan, and inhibited cell-cell fusion via a direct effect on the F0 protein, but did not show any virucidal effect. The mixture of ulvan and fucoidan showed a greater anti-spread effect than SPs alone, but ulvan antagonizes the effect of fucoidan on the viral attachment/entry. Both SPs may be promising antivirals against paramyxovirus infection but their mixture has no clear synergistic advantage.

Figure 1

Infrared absorption spectrum of the native ulvan from Ulva clathrata.

Figure 2

¹H-NMR spectrum in D₂O of the native ulvan from Ulva clathrata.

Figure 3

Cytotoxicity and antiviral activity of sulphated polysaccharides (SP) against Newcastle Disease Virus (NDV). (a) Vero cells (6.5 × 10³ cells), seeded in 96-well plates, were treated with different concentrations of SP for 48 h. Cell viability was then determined in triplicates by the MTT assay and compared with that in untreated cells (100% viability); (b) Vero cells (6.7 × 10⁴ cells), seeded in 24-well plates, were infected with NDV and treated with different concentrations of SP during infection for 1 h, and the antiviral activity was determined in a syncytia reduction assay. The data shown are the mean ± standard deviation (SD) of triplicate cultures, and the experiment was repeated three times. CC, cellular control; CV, viral control.

Figure 4

Dose-effect relationships of fucoidan and ulvan, alone or in combination. (a) A dose-effect curve based on data obtained in syncytia reduction assay, the curve depicts no sigmoidal (synergism) or plateau effect (summation) shapes indicating an antagonism relationship between the SP; (b) Combination Index (CI) values of the mixture of SP at different Effective Doses (ED) based on syncytia reduction assay and calculated by the median-effect method of Chou and Talalay. CI values > 1 indicates an antagonism effect; (c) A polygonogram plot indicating a very strong antagonism (very thick dash line) between the SP.

Figure 5

Inhibition of an avirulent NDV (La Sota) strain-mediated cell fusion. (A) Fusion inhibition assay with SP and their mixture treatment before, before cleavage [BC] and after F protein cleavage, after cleavage [AC]. Data are expressed as the percent of the number of syncytia compared with the findings in virus-infected control cells. VC, viral control; F, fucoidan; U, ulvan; M, mixture. The data shown are the mean ± SD of triplicate experiments. The asterisks indicate a significant difference between the treatment and viral control (* p < 0.05 and ** p < 0.001); (B) Vero cells infected with NDV and treated by trypsin digestion: (a) Uninfected control cells (100×); (b) Vero cells infected with NDV and treated with trypsin (100×); (c) Infected Vero cells were treated with 0.1 μg/mL of the SP and their mixture before F protein cleavage, at which point syncytia formation could be inhibited if SP were added (100×); (d) Infected Vero cells were treated with SP and their mixture after F protein cleavage, and the SP could not inhibit syncytia formation in this condition (100×).