Antiviral activity of lambda-carrageenan against influenza viruses and severe acute respiratory syndrome coronavirus 2

Abstract

Influenza virus and coronavirus, belonging to enveloped RNA viruses, are major causes of human respiratory diseases. The aim of this study was to investigate the broad spectrum antiviral activity of a naturally existing sulfated polysaccharide, lambda-carrageenan (λ-CGN), purified from marine red algae. Cell culture-based assays revealed that the macromolecule efficiently inhibited both influenza A and B viruses with EC₅₀ values ranging from 0.3 to 1.4 μg/ml, as well as currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an EC₅₀ value of 0.9 ± 1.1 μg/ml. No toxicity to the host cells was observed at concentrations up to 300 μg/ml. Plaque titration and western blot analysis verified that λ-CGN reduced expression of viral proteins in cell lysates and suppressed progeny virus production in culture supernatants in a dose-dependent manner. This polyanionic compound exerts antiviral activity by targeting viral attachment to cell surface receptors and preventing virus entry. Moreover, its intranasal administration to mice during influenza A viral challenge not only alleviated infection-mediated reductions in body weight but also protected 60% of mice from virus-induced mortality. Thus, λ-CGN could be a promising antiviral agent for preventing infection with several respiratory viruses.

Figure 1

Inhibition of influenza virus infection by λ-CGN in vitro. (A) Chemical structure of the repeating disaccharide unit in λ-CGN. (B) Western blot analysis showing expression of viral proteins. MDCK cells infected with PR8 at an MOI of 0.001 were mock-treated (Mock) or treated with 10 μM of OSV-C or with increasing concentrations of λ-CGN or p-KG03 at 35 °C. On the next day, cell lysates were harvested for SDS-PAGE and immunoblotting with anti-NP or anti-HA antibodies. β-Actin was used as a loading control. ‘No virus’ means negative control without viral infection. Proteins are indicated on the right side of the panels. (C) Plaque assay to determine viral titers. Serial ten-fold dilutions of cell culture supernatants acquired after viral infection and compound treatment in (B) were loaded onto fresh MDCK cells and cultured at 33 °C in 1.2% Avicel-containing overlay medium. The number of viral plaques was counted after crystal violet staining on day 3 post-infection. Data are expressed as the mean ± S.D. of three independent experiments. ****P < 0.0001. The graph was created using GraphPad Prism 8.3.1 (www.graphpad.com).

Figure 2

Time-of-addition experiment. (A) Schematic representation of compound treatment and virus infection. In the pre-treatment assay, MDCK cells were treated with λ-CGN (1 μg/ml) for 2 h and then infected with PR8 at an MOI of 0.001 for additional 2 h. In the co-treatment assay, the mixture of λ-CGN and PR8 was loaded onto the cells immediately or after 30-min-preincubation [Co-treatment (− 30 min)]. In the post-treatment assay, PR8-infected cells were added with λ-CGN for 2 h. As controls, p-KG03 (1 μg/ml) or EGCG (1 μM) was treated in all sets. In each step, cells were washed with PBS to remove non-specifically bound compounds or unabsorbed virus. (B) Plaque titration. The cells treated with compounds and PR8 at different time points were incubated in the overlay medium. At day 3 post-infection, viral plaques were counted in comparison to the mock-treated controls (set at 100%). Data are expressed as the mean ± S.D. of three independent experiments. *P < 0.05; **P < 0.01; ****P < 0.0001; n.d., not detected. Pre, pre-treatment of the compound; Co, co-treatment of the compound with virus; Co (− 30), co-treatment with 30 min-preincubated samples; post, post-treatment of the compound. The graph was created using GraphPad Prism 8.3.1 (www.graphpad.com).

Figure 3

Effect of λ-CGN on the influenza A virus entry. (A) HA inhibition assay. Two-fold serially diluted PR8 (from 2³ to 2¹¹) in PBS was incubated with an equal volume of PBS or twofold increasing concentrations of λ-CGN for 20 min. HA titer in each combination was determined at 30 min after addition of 0.5% chicken RBC. HA titers are marked on the right side of the panel. (B,C) Confocal microscopy. MDCK cells were infected with PR8 (MOI, 5) in the absence (Mock) or presence of either λ-CGN or p-KG03 at a concentration of 10 μg/ml. At 4 h post-infection in the absence of CHX (B) or at 2.5 h in the presence of 10 μg/ml CHX (C), viral NP was detected with an anti-NP antibody and an Alex Fluor 488-conjugated goat anti-mouse secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Original magnification, 400×. The images were analyzed using ZEN blue software 3.1 (www.zeiss.com).

Figure 4

Antiviral effect of λ-CGN against influenza A virus in vivo. BALB/c mice (6–7 weeks old female) were mock-infected (black) or intranasally infected with maPR8 at 5 MLD₅₀ (red). As test groups, the virus was preincubated at room temperature for 30 min with λ-CGN at a lower dose (1 mg/kg/d, purple) or a higher dose (5 mg/kg/d, green), followed by intranasal administration. Control mice received OSV-P orally twice a day (10 mg/kg/d) at 8-h intervals, starting at 4 h before viral infection (blue). Body weight (A) and mortality (B) of mice were measured every day from days 0 to 14 post-infection. Data are expressed as the mean ± S.D. from five mice. Survival statistics were calculated by Log-rank (Mantel–Cox) test. **P < 0.01; n.s., non-statistically significant. The graphs were created using GraphPad Prism 8.3.1 (www.graphpad.com).

Figure 5

Inhibition of infection by influenza A virus (H1N1) HA/NA- or SARS-CoV-2 spike-pseudotyped viruses. Lentiviral pseudotypes bearing influenza A virus (H1N1) HA and NA proteins (black bars) or SARS-CoV-2 spike protein (gray bars) were prepared, in which a firefly luciferase-expressing plasmid was incorporated. They were preincubated with mock or increasing concentrations of λ-CGN for 2 h at 37 °C and then infected into Vero E6 cells. On day 2, relative firefly luciferase activity (RLU) was determined by fixing the mock-treated sample at 100%. Data are expressed as the mean ± S.D. of three independent experiments. ****P < 0.0001. The graph was created using GraphPad Prism 8.3.1 (www.graphpad.com).

Figure 6

Image-based antiviral analysis of λ-CGN against SARS-CoV-2. (A) Vero cells seeded in 96-well plates were infected with SARS-CoV-2 at an MOI of 0.02, either alone or in the presence of increasing concentrations of λ-CGN (upper panel) or RDV (lower panel; a control). On day 2 post-infection, cells were fixed and permeabilized prior to immunostaining with an anti-SARS-CoV-2 spike antibody and an Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Cell nuclei were counterstained with DAPI to estimate cell viability (blue). Images were captured with a 20× objective lens fitted to an automated fluorescence microscope by using the Harmony High-Content Imaging and Analysis software 3.5.2 (www.perkinelmer.com). (B) The number of fluorescent green and blue spots was counted to calculate antiviral activity (red circles) and cell viability (black squares), respectively, at each concentration of the compounds. The viability of mock-infected cells were fixed as 100%, while the antiviral activity in virus-infected cells or mock-infected cells was fixed as 0 and 100%, respectively. Data are expressed as the mean ± S.D. from three independent experiments. The graphs were created using GraphPad Prism 8.3.1 (www.graphpad.com).

Figure 7

Anti-SARS-CoV-2 activity of λ-CGN. (A) SARS-CoV-2-infected Vero cells (MOI, 0.005) were treated with λ-CGN or RDV for 2 days. Whole cell lysates were subjected to immunoblotting with anti-SARS-CoV-2 spike antibody (upper panel), in which β-actin was used as a loading control (lower panel). Proteins are marked on the right side of the panels. No virus, cell lysates without SARS-CoV-2 infection; Mock, SARS-CoV-2-infected cell lysates without antiviral compound treatment. S2, S2 subunit of viral spike protein. (B) Viral RNA was purified from the culture supernatants of the samples as mentioned in (A). Real-time RT-PCR was conducted using SARS-CoV-2 N gene-specific primers. Relative viral RNA copies were calculated on the basis of their Cq values. (C) Culture supernatants used in (B) were serially diluted for infection of fresh Vero cells. On day 2, the cell culture plates were subjected to immunofluorescence assay for determination of infectious viral titer by calculating the relative ratio of spike-derived green fluorescence frequency to nuclei-derived blue-positive cell number. Data are expressed as the mean ± S.D. of three independent experiments. **P < 0.01; ****P < 0.0001; n.d., not detected. The graphs were created using GraphPad Prism 8.3.1 (www.graphpad.com).