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. 2020 Apr 17;177:113982. doi: 10.1016/j.bcp.2020.113982

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© 2020 Elsevier Inc. All rights reserved.

Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

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Fig. 3 — a Cytopathic effects (CPE) on MDCK cells infected with influenza A/PR/8/34 (H1N1) (100 TCID₅₀). Four different drug administration protocols were employed to treat the virus or cells at the concentration of 5 μg/mL of PPa. Phase contrast images were obtained using a microscope at 48 h post-infection. 3b Time-of-addition assay. MDCK cells infected with A/PR/8/34(H1N1) (100 TCID₅₀) were treated with 5 μg/mL PPa at the indicated time intervals. The virus titer was determined by measuring the TCID₅₀ of virus in the supernatant at 48 h post-infection; *p < 0.05, ^**p < 0.01, and ^***p < 0.001. 3c Hemagglutinin inhibition assay designed to measure the inhibitory effect of PPa on the binding of the virus to target cells. An equal volume of influenza A/Puerto Rico/8/34(H1N1) virus (4HAU) was transferred to a microwell plate containing two-fold serially diluted PPa in PBS. Then, erythrocytes (1% v/v in saline) were added to each well. After an incubation at room temperature for 1 h, the hemagglutination reaction was analyzed. PBS without virus served as a positive control, while virus served as the negative control. 3d Hemolysis inhibition assay. A suspension of freshly prepared chicken erythrocytes (2%) was added to the mixture of PPa (15 μg/mL) with the influenza virus A/PR/8/34 (H1N1) in allantoic fluid. The mixture was then acidified to a pH ranging from 4.6 to 6.0 (sodium acetate, 0.5 M), and incubated at 37 °C for 30 min. At the end of the incubation, the mixture was centrifuged and the absorbance of the supernatants containing released hemoglobin was measured at OD₅₃₅using a Multiskan FC microplate reader. 3e Neuraminidase (NA) inhibition assay. A reaction mixture consisting of PPa and influenza A/Puerto Rico/8/34(H1N1) virus in MES buffer was incubated for 45 min. Afterwards, 4-MU-NANA was added to each well, the mixture was incubated for another 1 h, and the reaction was terminated with NaOH (83% ethanol). Finally, an excitation wavelength of 340 nm and an emission wavelength of 440 nm were used to measure resulting fluorescence of the mixture. Oseltamivir phosphate and zanamivir were employed as positive controls.

Fig. 3 — a Cytopathic effects (CPE) on MDCK cells infected with influenza A/PR/8/34 (H1N1) (100 TCID₅₀). Four different drug administration protocols were employed to treat the virus or cells at the concentration of 5 μg/mL of PPa. Phase contrast images were obtained using a microscope at 48 h post-infection. 3b Time-of-addition assay. MDCK cells infected with A/PR/8/34(H1N1) (100 TCID₅₀) were treated with 5 μg/mL PPa at the indicated time intervals. The virus titer was determined by measuring the TCID₅₀ of virus in the supernatant at 48 h post-infection; *p < 0.05, ^**p < 0.01, and ^***p < 0.001. 3c Hemagglutinin inhibition assay designed to measure the inhibitory effect of PPa on the binding of the virus to target cells. An equal volume of influenza A/Puerto Rico/8/34(H1N1) virus (4HAU) was transferred to a microwell plate containing two-fold serially diluted PPa in PBS. Then, erythrocytes (1% v/v in saline) were added to each well. After an incubation at room temperature for 1 h, the hemagglutination reaction was analyzed. PBS without virus served as a positive control, while virus served as the negative control. 3d Hemolysis inhibition assay. A suspension of freshly prepared chicken erythrocytes (2%) was added to the mixture of PPa (15 μg/mL) with the influenza virus A/PR/8/34 (H1N1) in allantoic fluid. The mixture was then acidified to a pH ranging from 4.6 to 6.0 (sodium acetate, 0.5 M), and incubated at 37 °C for 30 min. At the end of the incubation, the mixture was centrifuged and the absorbance of the supernatants containing released hemoglobin was measured at OD₅₃₅using a Multiskan FC microplate reader. 3e Neuraminidase (NA) inhibition assay. A reaction mixture consisting of PPa and influenza A/Puerto Rico/8/34(H1N1) virus in MES buffer was incubated for 45 min. Afterwards, 4-MU-NANA was added to each well, the mixture was incubated for another 1 h, and the reaction was terminated with NaOH (83% ethanol). Finally, an excitation wavelength of 340 nm and an emission wavelength of 440 nm were used to measure resulting fluorescence of the mixture. Oseltamivir phosphate and zanamivir were employed as positive controls.