Oseltamivir

The neuraminidases from mammalian influenza virus PR8-H1N1 and avian influenza 3937-H6N1 were utilized to investigate the inhibition ability of VRE. The neuraminidase inhibition assay was performed according to Zhu et al. (2015) (link) with some modifications. Briefly, 1 μl of various concentrations of VRE or oseltamivir (Sigma) was mixed with 25 μl of virus (32 HAU of 3937-H6N1 virus or PR8-H1N1 virus) and 1× assay buffer (33 mM MES, 4 mM CaCl₂, pH 6.5) to a total of 50 μl and then incubated for 20 min at 37°C. Fifty microliters of fluorogenic substrate (50 μM 4-methylumbelliferyl-N-acetylneuraminic acid, Sigma) was added and incubated for 60 min at 37°C. The reaction was then terminated by adding 100 μl of 0.2 M Na₂CO₃. Fluorescence was measured with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Relative fluorescence units were obtained by subtracting the background value.

Mitoxantrone, rhodamine 123, GF120918 (Elacridar), Ko143, hydrocortisone, prednisolone, dexamethasone, ivermectin, lopinavir, hydroxychloroquine, chloroquine and oseltamivir and MTT were purchased from Sigma-Aldrich. Hoechst 33342 and TRIzol were purchased from Invitrogen. All other reagents were commercial products of the highest available purity.

MDCK cells (2 × 10⁵/mL) were cultured in 96-well plates. The cells were infected with 200 TCID₅₀ of A/PR/8 H1N1 influenza virus. After virus absorption for 1 h at 35 °C, the medium containing PP was added in each well and the plate was incubated at 35 °C and 5% CO₂. CPE was observed daily and the results were read after 4 days. Oseltamivir (Sigma-Aldrich, Milan, Italy) was used as positive control (5 µg/mL in water). Also, this test was performed in four replicates. The concentration of PP capable of inhibiting the viral growth in all the replicates was considered as having antiviral activity.

Six-week-old C57BL/6J mice were treated with 20 mg/kg AG1478 via laryngeal aspiration 3 h before IAV infection (1 × 10⁴ A/WSN/33 per mouse). The mice were given a second dose of AG1478 on the 2nd day and tissues were harvested on the 3rd day. Mouse lungs were homogenized followed by centrifugation. Supernatants were subject to plaque assay as described above. The control mice were given a vehicle control (DMSO). In a separate experiment, oseltamivir (Sigma-Aldrich, St. Louise, MO, USA) was used as a positive control and given to the mice at 20 mg/kg via the same laryngeal aspiration following the same protocol as AG1478. Ethics Statement: Mice used in the present study were covered under the animal protocol (#13-479) that was approved by the Institution Animal Care and Use Committee (IACUC) of the University of Arizona. Mice used in this study were housed in a barrier facility at the University of Arizona. The facility is accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC) and meets the requirements of the law and NIH regulations. All mice are routinely screened for a panel of pathogens and are cared for by certified animal care professionals. All the animal handling and experimental procedures adhere strictly to the general guidelines of the Animal Welfare Act (AWA), AAALAC and IACUC.

Virus isolates were produced as described above. Oseltamivir was purchased from Sigma Aldrich [Cat# SML1606]. CR8020 A/H3N2 stem antibody was kindly provided by Dr. Dirk Eggink and expressed following previously published procedures^{48 (link)}. Goat anti-human Alexa-647 [Cat# A21445] and streptavidin-AlexaFluor 635 [Cat# SA1011] antibodies were obtained from Thermo Fisher. Control lectins Erythrina cristagalli agglutinin (ECA) [Cat#B-1145], Sambuca nigra agglutinin (SNA) [Cat# B-1305], and Maackia amurensis lectin I (Mal-I) [Cat# B-1315] were purchased from Vector Labs.

The powdered zinc oxide nanoparticles were purchased from US Research Nanomaterials (USA; Product Number: US3590). Nanoparticles were suspended in Dulbecco’s Modified Eagle’s medium (DMEM) (Invitrogen, USA) and the suspension were subjected to sonication to prevent agglomeration and make different concentrations. Oseltamivir purchased from Sigma-Aldrich (St. Louis, MO, USA) were dissolved in DMEM and used as a standard drug against influenza at different concentrations. Polyethylene glycol (PEG) 6000 was also purchased from Sigma-Aldrich. PEGylated ZnO-NPs were synthesized by mechanical method, as described in detail previously [16 (link)]. Inductively coupled plasma mass spectrometry (ICP-MS), X-ray diffraction analysis (XRD), Transmission Electron Microscopy (TEM), and Field Emission Scanning Electron Microscope (FE-SEM) were used for characterization of nanoparticles. Thermogravimetric analysis (TGA) was also performed to demonstrate the presence of PEG on the surface of ZnO nanoparticles [16 (link)].