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. 2010 Oct;88(1):1-9.
doi: 10.1016/j.antiviral.2010.06.014. Epub 2010 Jul 14.

Influenza Virus Variation in Susceptibility to Inactivation by Pomegranate Polyphenols Is Determined by Envelope Glycoproteins

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Influenza Virus Variation in Susceptibility to Inactivation by Pomegranate Polyphenols Is Determined by Envelope Glycoproteins

Aarthi Sundararajan et al. Antiviral Res. .
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Abstract

Pomegranates have high levels of polyphenols (PPs) and may be a rich source of compounds with antiviral activity. We evaluated the direct anti-influenza activity of three commercially available pomegranate extracts: pomegranate juice (PJ), a concentrated liquid extract (POMxl), and a 93% PP powder extract (POMxp). The acidity of PJ and POMxl solutions contributed to rapid anti-influenza activity, but this was not a factor with POMxp. Studies using POMxp showed that 5min treatment at room temperature with 800μg/ml PPs resulted in at least a 3log reduction in the titers of influenza viruses PR8 (H1N1), X31 (H3N2), and a reassortant H5N1 virus derived from a human isolate. However, the antiviral activity was less against a coronavirus and reassortant H5N1 influenza viruses derived from avian isolates. The loss of influenza infectivity was frequently accompanied by loss of hemagglutinating activity. PP treatment decreased Ab binding to viral surface molecules, suggesting some coating of particles, but this did not always correlate with loss of infectivity. Electron microscopic analysis indicated that viral inactivation by PPs was primarily a consequence of virion structural damage. Our findings demonstrate that the direct anti-influenza activity of pomegranate PPs is substantially modulated by small changes in envelope glycoproteins.

Figures

Fig. 1
Fig. 1
Antiviral activity of PJ and POMxl against influenza virus. Influenza X31 (H3N2) at 5 × 107 TCID50/ml was treated for 5 min at room temperature with different concentrations of PJ (A) or POMxl (B), or with a pH-matched buffer. Infectious virus titers after treatment were determined by TCID50 assay. Results are shown as the fold reduction in titer relative to untreated virus (incubated with PBS). Data are mean ± SD for 3–6 separate experiments. *P < 0.01.
Fig. 2
Fig. 2
Antiviral activity of POMxp against influenza viruses X31 and PR8 and the coronavirus MHV A59. Influenza X31 (H3N2) (A) and PR8 (H1N1) (B) at 5 × 107 TCID50/ml were treated for 5 min at room temperature with different concentrations of POMxp (expressed as PP concentration). Infectious virus titers measured by TCID50 assay (filled squares, left axis) and HAg titers (open squares, right axis) were determined after treatment. MHV A59 (C) at 2.5 × 107 PFU/ml was treated with POMxp as for influenza virus and infectious virus titers were determined by plaque assay. Infectious virus titers are mean ± SD for 3–4 separate experiments. HAg titers are representative of three separate experiments that gave similar results.
Fig. 3
Fig. 3
Antiviral activity of POMxp against reassortant H5N1 and H1N1 influenza viruses and influenza NC/99 (H1N1). The following influenza viruses at the indicated titers were treated for 5 min at room temperature with different concentrations of POMxp (expressed as PP concentration): rg-VN/04 (H5N1) at 5 × 107 TCID50/ml (A), rg-Dk/HN/02 (H5N1) at 5 × 107 TCID50/ml (B), rg-Dk/LS/02 (H5N1) at 2.8 × 107 TCID50/ml (C), rg-JWE/HK/06 (H5N1) at 1 × 107 TCID50/ml (D), NC/99 (H1N1) at 2.5 × 105 TCID50/ml (E), and rg-CA/09 (H1N1) at 7.4 × 105 TCID50/ml. Infectious virus titers measured by TCID50 assay (filled squares, left axis) and HAg titers (open squares, right axis) were determined after treatment. Results are representative of at least two separate experiments that gave similar results.
Fig. 4
Fig. 4
Inhibition of virus-specific Ab binding after POMxp treatment. ELISA plates coated with the influenza viruses X31 (A), rg-VN/04 (B), or rg-Dk/HN/02 (C), or with MHV A59 (D) were incubated with serial dilutions of POMxp (expressed as PP concentration) prior to the addition of Abs specific for the coating Ags (mAbs specific for influenza H3 (X31 14-4), N2 (GY-14), and H5 (VN-10), and a rabbit antiserum (Vu-14) raised against MHV A59). Color development reflecting Ab binding was generated with enzyme-conjugated secondary Abs and substrate. The analysis was also performed using plates coated with influenza PR8 (H1N1) to control for non-specific adherence of Abs. Each point represents the mean ± SD for triplicate wells. Results are representative of two separate experiments that gave similar results.
Fig. 5
Fig. 5
Electron microscopic analysis of viruses after treatment with POMxp. Influenza viruses X31 (A and B), PR8 (C and D), and rg-Dk/HN/02 (E and F), and the coronavirus MHV A59 (G and H) at concentrations of 1 × 108 to 1 × 109 infectious units/ml were incubated for 5 min at room temperature with PBS (left-hand panels) or 1600 μg/ml PPs (right-hand panels).

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