y 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 -carrageenan preferentially targets adsorption steps. No difference was observed between the pretreatment-virus and adsorption group, suggesting that -carrageenan did not inactivate viral particles. 
In 946128-88-7 addition, the number of NP-positive cells in the IFA assay and comparison of mRNA levels confirmed this deduction. To further assess the inhibitory effect of -carrageenan on adsorption and internalization, the viral RNA levels were detected in groups treated with or without -carrageenan during adsorption and internalization. Fig 4D shows that the vRNA levels of NP in both adsorption and internalization groups treated with -carrageenan were significantly lower than in control groups. Taken together, these results suggest that -carrageenan had inhibitory actions directly on viral adsorption via target the viral particles instead of cells receptor, and internalization. -carrageenan specifically inhibited SW731 binding to its sialic acid receptor According to the data shown above, -carrageenan interfered with adsorption. It was here determined whether -carrageenan could inhibit the binding of HA to its receptors, Neu5Aca26Gal-terminated sugar chains. The HA titers of SW731 were markedly reduced by -carrageenan in a dose-dependent manner. To assess the specificity of this inhibitive effect, hemagglutination inhibition assays were also performed on several other influenza viruses. No difference in -carrageenan-treated virus PR8, WSN, ZB07, or LY08 was detected in response to PBS treatment, but -carrageenan can significantly decrease the HA titer of CA04, which contained a HA sharing 99% amino acid sequence with SW731. HA binding of SW26, which has a HA close to SW731 in evolutionary distance, was also slightly blocked by -carrageenan at concentration of 500 g/ml. These data together indicate that -carrageenan specifically inhibited HA of SW731 binding to its sialic acid receptor. Considering that -carrageenan may interact specifically with glycans on the HA protein, we analyzed the potential glycosylation site patterns on HA of 7 strains. As shown in 8 / 16 Carrageenan Specially Target HA of H1N1/2009 Virus Fig 4. -carrageenan-induced inhibition of viral adsorption and internalization. MDCK cells were infected with SW731 under 4 different treatment conditions. Pretreatment-SW731: SW731 was mixed with 250 g/ml of -carrageenan at 4C for 1 h before adsorption. Then the virus/ compound mixture was PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783938 added to cells for 1 h at 4C, and the media were removed and washed with PBS for three times to remove the compound. The cells were maintained in infective media at 37C for 24h; Pretreatment-cells: MDCK cells were treated with 250 g/ml of -carrageenan at 37C for 1 h before infection. After removing the compound, cells were washed with PBS and incubated with virus for 1 h at 4C. Then cells were maintained in infective media at 37C PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 for 24h; Adsorption: cells were incubated with virus/carrageenan mixture for 1 h of adsorption at 4C. Then cells were washed and overlaid with infective media at 37C for 24 h; After-adsorption: cells were incubated with virus for 1 h at 4C. After removal of unabsorbed virus, cells were overlaid 9 / 16 Carrageenan Specially Target HA of H1N1/2009 Virus with infective media containing 250 g/ml of -carrageenan fo