Antiviral effect of methylated flavonol isorhamnetin against influenza

Abstract

Influenza is an infectious respiratory disease with frequent seasonal epidemics that causes a high rate of mortality and morbidity in humans, poultry, and animals. Influenza is a serious economic concern due to the costly countermeasures it necessitates. In this study, we compared the antiviral activities of several flavonols and other flavonoids with similar, but distinct, hydroxyl or methyl substitution patterns at the 3, 3', and 4' positions of the 15-carbon flavonoid skeleton, and found that the strongest antiviral effect was induced by isorhamnetin. Similar to quercetin and kaempferol, isorhamnetin possesses a hydroxyl group on the C ring, but it has a 3'-methyl group on the B ring that is absent in quercetin and kaempferol. Co-treatment and pre-treatment with isorhamnetin produced a strong antiviral effect against the influenza virus A/PR/08/34(H1N1). However, isorhamnetin showed the most potent antiviral potency when administered after viral exposure (post-treatment method) in vitro. Isorhamnetin treatment reduced virus-induced ROS generation and blocked cytoplasmic lysosome acidification and the lipidation of microtubule associated protein1 light chain 3-B (LC3B). Oral administration of isorhamnetin in mice infected with the influenza A virus significantly decreased lung virus titer by 2 folds, increased the survival rate which ranged from 70-80%, and decreased body weight loss by 25%. In addition, isorhamnetin decreased the virus titer in ovo using embryonated chicken eggs. The structure-activity relationship (SAR) of isorhamnetin could explain its strong anti-influenza virus potency; the methyl group located on the B ring of isorhamnetin may contribute to its strong antiviral potency against influenza virus in comparison with other flavonoids.

Fig 1. Flavonoid chemical structure, impact on cell viability, post-treatment, pre-treatment, and co-treatment effects of the flavonoids against the influenza A/PR/8/34 (H1N1) virus.

(A) The chemical structures of flavonoids used in this study, showing different distributions of hydroxyl groups located on the B ring. (B) The experimental protocols for checking the antiviral activity of the tested flavonoids, which were as follows: pre-treatment, in which isorhamnetin and quercetin were administered before virus infection in a time-dependent manner (1 hr, 12 hr, and 24 hr) and a dose-dependent manner (10 μM, 50 μM, and 100 μM). Co-treatment, in which isorhamnetin or quercetin were incubated with the virus for different lengths of time during the incubation (0.5 hr, 1 hr, and 2 hr) and in a dose-dependent manner (10 μM, 50 μM, and 100 μM) before inoculation into the cells. Post-treatment, in which the flavonoids were administered after the end of the virus infection and removal of the virus.

Fig 2. Effect of isorhamnetin treatment on the virus-induced autophagy.

(A) Fluorescent microscope data for AVOs staining. MDCK cells were infected with the virus and then treated with isorhamnetin and finally stained with vital stains specific for AVOs (AO and MDC staining). (B) Western blot analysis for detection of LC3B protein lipidation. After infection with influenza A virus and treatment with isorhamnetin, we checked the lipidation of LC3B in a time-dependent manner (6 hr, 9 hr, and 12 hr). (C)RT-PCR data showing the expression level of autophagy genes. After virus infection and isorhamnetin treatment, we checked the expression level of autophagy related genes (Atg-5, Atg-7, and LC3B) using RT-PCR. Loading control = GAPDH.

Fig 3. Measurement of antiviral activity of isorhamnetin by virus yield and HI assays.

(A) Viral yield reduction assay was carried out by seeding MDCK cells in a 6-well plate infected with influenza A/PR/8/34 (H1N1) viruses for 2 hr, followed by virus removal, and flavonoids treatment in a dose dependent manner (10 μM, 50 μM, and 100 μM) for 48 hr. After incubation, a virus yield reduction assay was carried out using media soup. The HA titers were interpreted as HAU/50μL. *P < 0.05, **P <0.01. (B) Hemagglutination inhibition (HI) assay: The flavonoids were serially diluted using PBS and were added to an equal volume of the virus. For checking RBCs hemolysis inhibition potency, 50μL of 1% chicken RBCs were added to each well of a 96-well plate incubated for 30min at room temperature. *P < 0.05, **P <0.01.

Fig 4. Measurement of antiviral activity of isorhamnetin by NA assay and RT-PCR.

(A) NAI assay was assessed by mixing of 50 μL of the flavonoids at the indicated concentrations with 50 μL of the virus, and then adding 100μL of the substrate solution (4-MU-NANA; (2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminicacid, sodium salt hydrate; Sigma-Aldrich) that dissolved in the enzyme buffer [33mM 2-(N-morpholino) ethanesulfonic acid (MES), pH 6.5, and 4 mM CaCl2].This reaction mixture was protected from light and incubated at 37°C for 2hr under shaking condition. The optical density was measured for calculating the fluorescence intensity of 4-methylumbelliferone using fluorescence spectrophotometer at excitation of 365 nm and emission at 460 nm. *P < 0.05, **P <0.01, ***P <0.001. (B) RT-PCR was carried out to detect the expression level of HA and NA genes. MDCK cells were seeded in a 6-well plate infected with influenza A/PR/8/34 (H1N1) viruses for 2 hr, followed by virus removal and flavonoids treatment in a dose dependent manner (10 μM, 50 μM, and 100 μM) for 48 hr. After incubation, RT-PCR was performed using specific primers for influenza virus HA and NA.

Fig 5. Effect of isorhamnetin on the inhibition of the influenza virus-induced ROS generation and ERK phosphorylation.

(A) After influenza virus infection and isorhamnetin treatment, ROS generation was measured using ROS fluorescent probe, H₂DCFDA. Scale bar = 200 μm. (B) Western blot analysis for phosphorylation of ERK. After influenza A virus infection and isorhamnetin treatment, we checked the phosphorylation level of ERK in a time-dependent manner (6 hr, 9 hr, and 12 hr) after virus infection (post-treatment).

Fig 6. In vivo antiviral activity of isorhamnetin against influenza virus infection.

(A)The rate of loss in body weight of six-week-old female mice(C57BL/6)after influenza A/PR/8/34 (H1N1) virus infection and isorhamnetin treatment, and Tamiflu was used as a positive anti-influenza material; *P < 0.05. (B) The survival rate in six-week-old female mice (C57BL/6) after influenza A/PR/8/34 (H1N1) virus infection and flavonoids treatment. *P < 0.05.

Fig 7. Schematic diagram represents the detailed mechanism of isorhamnetin in its antiviral activity against influenza A virus.

Isorhamnetin possesses potent direct or indirect anti-influenza activity via direct suppression of virus adsorption onto host cells (HI) and NA activity (NAI) or indirect inhibition of the expression of influenza A surface proteins (HA and NA), virus-induced ROS generation and ERK phosphorylation, and the autophagic changes (AVOs formation and lipidation of LC3B) after influenza A virus infection.