Specific Inhibitory Effect of κ-Carrageenan Polysaccharide on Swine Pandemic 2009 H1N1 Influenza Virus

Abstract

The 2009 influenza A H1N1 pandemic placed unprecedented demands on antiviral drug resources and the vaccine industry. Carrageenan, an extractive of red algae, has been proven to inhibit infection and multiplication of various enveloped viruses. The aim of this study was to examine the ability of κ-carrageenan to inhibit swine pandemic 2009 H1N1 influenza virus to gain an understanding of antiviral ability of κ-carrageenan. It was here demonstrated that κ-carrageenan had no cytotoxicity at concentrations below 1000 μg/ml. Hemagglutination, 50% tissue culture infectious dose (TCID50) and cytopathic effect (CPE) inhibition assays showed that κ-carrageenan inhibited A/Swine/Shandong/731/2009 H1N1 (SW731) and A/California/04/2009 H1N1 (CA04) replication in a dose-dependent fashion. Mechanism studies show that the inhibition of SW731 multiplication and mRNA expression was maximized when κ-carrageenan was added before or during adsorption. The result of Hemagglutination inhibition assay indicate that κ-carrageenan specifically targeted HA of SW731 and CA04, both of which are pandemic H1N/2009 viruses, without effect on A/Pureto Rico/8/34 H1N1 (PR8), A/WSN/1933 H1N1 (WSN), A/Swine/Beijing/26/2008 H1N1 (SW26), A/Chicken/Shandong/LY/2008 H9N2 (LY08), and A/Chicken/Shandong/ZB/2007 H9N2 (ZB07) viruses. Immunofluorescence assay and Western blot showed that κ-carrageenan also inhibited SW731 protein expression after its internalization into cells. These results suggest that κ-carrageenan can significantly inhibit SW731 replication by interfering with a few replication steps in the SW731 life cycles, including adsorption, transcription, and viral protein expression, especially interactions between HA and cells. In this way, κ-carrageenan might be a suitable alternative approach to therapy meant to address anti-IAV, which contains an HA homologous to that of SW731.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Cytotoxic effect of κ-carrageenan on MDCK cells.

MDCK cells were treated with the indicated concentrations of κ-carrageenan. After 48 h of incubation, metabolic activity was measured via MTT assay. Results were analyzed with the independent sample t test (n = 3). Values are means ± SEM (n = 3). Significance: *P < 0.05 vs. nondrug-treated control group; **P < 0.005 vs. nondrug-treated control group. Results are representative of two independent experiments.

Fig 2. Anti-SW731 effect of κ-carrageenan on MDCK cells.

(A, B) MDCK cells were incubated with SW731, CA04, PR8, WSN, and ZB07 at 4°C (MOI = 0.1) and then treated with κ-carrageenan or Ribavirin at the indicated concentration after removal of the virus inoculum. After 24 h, the viral titers were determined by HA and TCID50 assays. (C, D) MDCK cells were incubated with SW731, CA04, PR8, WSN, and ZB07 at 4°C (MOI = 1) and then treated with κ-carrageenan or Ribavirin at the indicated concentration after removal of the virus inoculum. After 24 h, the viral titers were determined by HA and TCID50 assays. (E) MDCK cells were infected with SW731 at an MOI of 1 and then treated with κ-carrageenan at the indicated concentration after removal of the viral inoculum. After 48 h, CPE inhibition was determined by CPE assay. Results were analyzed with the independent sample t test (n = 3). Values are means ± SEM. Significance: *P < 0.05 vs. nondrug treated control group; **P < 0.005 vs. nondrug treated control group. Results are representative of two independent experiments.

Fig 3. Time of addition of κ-carrageenan on SW731 replication.

MDCK cells were infected with SW731 at an MOI of 3. κ-carrageenan was added to the cells at 0 h (adsorption), 1 h, 2 h, 4 h, 6 h, and 8 h. After 24 h, viral titers were determined using TCID50 assays. Results were analyzed with the independent sample t-test (n = 3). Values are means ± SEM. Significance: *P < 0.05 vs. nondrug treated control group; **P < 0.005 vs. nondrug treated control group. $P < 0.05 vs. 8 h group. Results are representative of two independent experiments.

Fig 4. κ-carrageenan-induced inhibition of viral adsorption and internalization.

(A–C) MDCK cells were infected with SW731 (MOI = 3) under 4 different treatment conditions. (i) Pretreatment-SW731: SW731 was mixed with 250 μg/ml of κ-carrageenan at 4°C for 1 h before adsorption. Then the virus/compound mixture was added to cells for 1 h at 4°C, and the media were removed and washed with PBS for three times to remove the compound. The cells were maintained in infective media at 37°C for 24h; (ii) Pretreatment-cells: MDCK cells were treated with 250 μg/ml of κ-carrageenan at 37°C for 1 h before infection. After removing the compound, cells were washed with PBS and incubated with virus for 1 h at 4°C. Then cells were maintained in infective media at 37°C for 24h; (iii) Adsorption: cells were incubated with virus/carrageenan mixture for 1 h of adsorption at 4°C. Then cells were washed and overlaid with infective media at 37°C for 24 h; (iiii) After-adsorption: cells were incubated with virus for 1 h at 4°C. After removal of unabsorbed virus, cells were overlaid with infective media containing 250 μg/ml of κ-carrageenan for 1h at 37°C. Then cells were washed with PBS and maintained in compound free infective media for 24 h. Viral titers were determined in a series of TCID50 assays. The mRNA levels and NP-positive cells were detected using qRT-PCR and immunofluorescence staining, respectively. (D) MDCK cells were infected with SW731 (MOI = 3) by 2 different treatment conditions. (i) Adsorption: MDCK cells were incubated with SW731 in the presence or absence (control) of κ-carrageenan at concentration of 250 μg/ml. One hour later, the media were removed and total RNA was isolated. (ii) Internalization: MDCK cells were incubated with SW731 at 4°C for 1 h. After removal of the inoculation, the cells were overlaid with infective media in the presence or absence of κ-carrageenan at concentration of 250 μg/ml at 37°C for 1 h, after treatment with protease K for 5 min, total RNA was extracted and analyzed using qRT-PCR. Results were analyzed using the independent sample t test. Values are means ± SEM (n = 3). Significance: *P < 0.05 vs. nondrug treated control group; **P < 0.005 vs. nondrug treated control group; #P < 0.05 vs. after adsorption group. Results are representative of two independent experiments.

Fig 5. Specific inhibition effect of κ-carrageenan on SW731 HA.

(A–C) Twenty-five microliters of serially diluted SW731, SW26, CA04, PR8, WSN, LY08, and ZB07 viruses were incubated with the same volume of PBS (Mock) or serially diluted κ-carrageenan, at RT for 20 min. Then each sample was mixed with 50 μl of 1% chicken red blood cells (RBC). After 30 min, the concentrations of κ-carrageenan inhibiting HA activity were measured. (D) Phylogenetic trees of the HA genes were constructed with sequences from GenBank based on the open reading frame sequences. The tree was generated using the neighbor-joining method in MEGA 6.0, with 1000 bootstrap trials performed to assign confidence to the grouping. Results were analyzed with the independent sample t test (n = 3). Values are means ± SEM. Significance: *P < 0.05 vs. nondrug treated control group. **P < 0.005 vs. nondrug treated control group. Results are representative of two independent experiments.

Fig 6. Inhibition effect of κ-carrageenan on SW731 mRNA and protein expression.

(A) MDCK cells were infected with SW731 (MOI = 3) for 1h, and then treated with 250 μg/ml of κ-carrageenan or PBS after virus internalization. After 6 h, total RNA was extracted and analyzed using qRT-PCR. (B) MDCK cells were infected with SW731 (MOI = 3), and then treated with 250 μg/ml of κ-carrageenan or PBS after virus internalization. After 8 h, Western blot was performed using anti-NP protein antibody and HRP-labeled second antibody. (C) MDCK cells were infected with SW731 (MOI = 3), and then treated with 250 μg/ml of κ-carrageenan or PBS after viral internalization. After 8 h, immunofluorescence staining was performed using anti-NP protein antibody and FITC-labeled second antibody, and NP positive cell percentage was measured. Results were analyzed with the independent sample t test (n = 3). Values are means ± SEM. Significance: *P < 0.05 vs. nondrug treated control group; **P < 0.005 vs. nondrug treated control group. Results are representative of two independent experiments.