Oseltamivir

Neuraminidase activity and oseltamivir (F. Hoffmann–La Roche Ltd., Basel, Switzerland) susceptibility of the strains A/43, A/324, A/962, A/1377-Amer, A/1017, and A/999 were determined using a fluorescent neuraminidase inhibition (NAI) assay according to a previously described method. Briefly, viruses were standardized to an NA activity level 10-fold higher than that of the background, as measured by the production of a fluorescent product from 20-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid substrate (MUNANA; Sigma-Aldrich, Darmstadt, Germany). Drug susceptibility profiles were determined by the extent of NA inhibition after incubation with three-fold serial dilutions of NAIs. The 50% inhibitory concentrations (IC_50s) were determined from the dose–response curve. The enzymatic reaction was read with a Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA.) microplate reader with excitation and emission wavelengths of 360 and 460 nm, respectively. This work involved the use of equipment from the multiaccess center “Modern Optical Systems” of the Federal Research Center of Fundamental and Translational Medicine (Novosibirsk, Russia). All methods were performed in accordance with the relevant guidelines and regulations.

Specific-pathogen-free (SPF) C57BL/6 mice (6–8-week-old males, n = 90) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., (beijing, China). Mice were randomly assigned to three groups: the H5N1 virus infection group (vehicle, treated with PBS, n = 45), oseltamivir (OS, treated with oseltamivir, n = 45), and geldanamycin (GA, treated with geldanamycin, n = 45). Mice were intranasally infected with 1 × 10⁵ pfu (equivalent to 10 LD₅₀) of the mouse adapted influenza virus A/environment/Qinghai/1/2008 (H5N1) under anesthesia with isoflurane. Two hours after infection, mice in the experimental groups were administered 5 mg/kg of OS (oseltamivir, twice daily for 5 days) (Roche, Switzerland) in PBS by gavage (Sidwell et al., 1998 (link)) or 1 mg/kg of GA (geldanamycin, twice daily for 5 days) (NCPC, CHN) in DMSO (dimethyl sulfoxide) (Sigma-Aldrich, USA) by intraperitoneal injection (Chatterjee et al., 2007 (link); Nakano et al., 2007 (link)), whereas mice in the control group received PBS only. Body weights and survival of each group were monitored for 12 days or until death. All experiments with influenza virus infections were conducted in a Biological Safety Level-3 (BSL-3) laboratory. Food and water were available ad libitum.

Highly pathogenic influenza A/Turkey/15/2006 (H5N1) virus (HA clade 2.2.1) was propagated in 10-day-old embryonated chicken eggs for 48 h at 35 °C. Influenza A/Turkey/15/2006 (H5N1) virus was well-characterized previously43 (link) and represents clade 2, the HA clade of A(H5N1) viruses that infects humans and continues to evolve rapidly. Madin-Darby canine kidney (MDCK) cells (ATCC, Manassas, VA) were maintained in Eagle’s minimum essential medium (MEM) (Gibco) supplemented with 5% fetal calf serum. The prodrug Oseltamivir phosphate (Oseltamivir) was dissolved in sterile distilled water, and T-705 was resuspended in ORA-PLUS suspending vehicle (Paddock laboratories, LLC). Oseltamivir and T-705 were provided by F. Hoffmann-La Roche Ltd. (Basel, Switzerland).

The co-infections were performed as previously described [9 (link)]. Briefly, the MDCK cells were first infected with H1N1pdm09 at a multiplicity of infection (MOI) of 0.5 for 1 h at 34 °C. The cells were washed and incubated for 3 additional h at 34 °C in EMEM containing 1 µM oseltamivir (kindly provided by Hoffmann-La Roche, Ltd., Basel, Switzerland). The cells were then washed and infected with H5TK13 at a MOI of 0.5 for 1 h at 34 °C. The cells were finally washed and cultured for 18 h at 34 °C in EMEM containing 1 µM oseltamivir. The progeny viruses were then plaque purified: 10-fold serial dilutions of supernatants were used to inoculate the MDCK cells for 1 h at 34 °C, and then, the cells were overlaid with EMEM containing 0.55% agar. After 48 h at 34 °C, 50 viral clones were isolated, propagated in the MDCK cells, and stored at −80 °C. The genotyping was performed by Sanger sequencing of the cDNA amplified with sets of primers identifying the gene origins [10 (link),11 (link)].

Susceptibility of viral NA to oseltamivir (Roche Diagnostics GmbH, Mannheim, Germany) and zanamivir (GlaxoSmithKline, Uxbridge, UK) was assessed by fluorescent neuraminidase activity inhibition [17 (link)]. The NA activity was measured using the fluorescent substrate, 2’-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA; Sigma, USA) and the inhibitor concentrations ranged from 0.03 nmol/L to 1,000 nmol/L. Briefly, a total volume of 45 μL containing 15 μL of the virus and 66 μM MUNANA in 21.66 mM MES buffer (pH 6.5) containing 2.66 mM CaCl₂ was incubated for 60 minutes at 37 °C, and the reaction was stopped by addition of 150 μL of 0.14 M NaOH in 83% ethanol. The fluorescence of the released 4-methylumbelliferone sodium salt was measured at excitation and emission wavelengths of 355/365 nm and 450/460 nm, respectively. The activity of each virus sample was titrated, by assaying serial two-fold dilutions. Virus suspensions were adjusted to equivalent NA activities, which fell into the linear portion of the activity curve and the viruses were pre-incubated for 30 min at 37 °C with oseltamivir or zanamivir at final concentrations of 5 μM to 0.05 pM, in serial ten-fold dilutions.