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

Shao Q, Guo Q, Xu Wp, Li Z, Zhao Tt - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, No. 2 Yuan Ming Yuan West Road, Beijing, 100193, China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0126577.g004: 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.

Mentions: To further elucidate the mechanism underlying the anti-SW731 activity of κ-carrageenan, we also investigated whether κ-carrageenan elicits its inhibitory actions directly on virus particles, cell receptor, viral adsorption or internalization. MDCK cells were treated with κ-carrageenan at four different conditions, pretreated-SW731, pretreated-cells, during adsorption, and after-adsorption. After 24 h viral titers in the culture media were determined by TCID50 assay. Fig 4A shows that pretreated-SW731, SW731 during adsorption, and SW731 after adsorption could all significantly decrease the viral titer in all samples except pretreated cells, suggesting that κ-carrageenan could interfere with binding of SW731 and MDCK by targeting the virus instead of MDCK cells, and blocking internalition. In other cases, both pretreatment-virus and adsorption groups showed significantly lower viral titers than the after-adsorption group, indicating that κ-carrageenan preferentially targets adsorption steps. No difference was observed between the pretreatment-virus and adsorption group, suggesting that κ-carrageenan did not inactivate viral particles. In addition, the number of NP-positive cells in the IFA assay and comparison of mRNA levels confirmed this deduction (Fig 4B and 4C).


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

Shao Q, Guo Q, Xu Wp, Li Z, Zhao Tt - PLoS ONE (2015)

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.
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pone.0126577.g004: 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.
Mentions: To further elucidate the mechanism underlying the anti-SW731 activity of κ-carrageenan, we also investigated whether κ-carrageenan elicits its inhibitory actions directly on virus particles, cell receptor, viral adsorption or internalization. MDCK cells were treated with κ-carrageenan at four different conditions, pretreated-SW731, pretreated-cells, during adsorption, and after-adsorption. After 24 h viral titers in the culture media were determined by TCID50 assay. Fig 4A shows that pretreated-SW731, SW731 during adsorption, and SW731 after adsorption could all significantly decrease the viral titer in all samples except pretreated cells, suggesting that κ-carrageenan could interfere with binding of SW731 and MDCK by targeting the virus instead of MDCK cells, and blocking internalition. In other cases, both pretreatment-virus and adsorption groups showed significantly lower viral titers than the after-adsorption group, indicating that κ-carrageenan preferentially targets adsorption steps. No difference was observed between the pretreatment-virus and adsorption group, suggesting that κ-carrageenan did not inactivate viral particles. In addition, the number of NP-positive cells in the IFA assay and comparison of mRNA levels confirmed this deduction (Fig 4B and 4C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, No. 2 Yuan Ming Yuan West Road, Beijing, 100193, China.

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.

No MeSH data available.


Related in: MedlinePlus