Neuraminidase Assay
Neuraminidase activity was measured using a commercially available chemiluminescent kit, NA-Star (Applied Biosystems, Foster City, CA, USA), which includes a chemiluminescent substrate, 1,2-dioxetane derivative of sialic acid (sodium(2chloro-5-(4-methoxyspiro1,2-dioxetane-3,29-(5-chloro)tricyclo [3.3.1.13,7]decan-4-yl-phenyl-5-acetamido-3,5-dideoxy-a-D-glycero-D-galacto-2-nonulopyranoside)onate), following the manufacturer’s protocol with minor modifications. A volume of 10 ml of sample dilutions and 40 ml of NA-star assay buffer were added to each well of a 96-well plate. Then 10 ml of 0.01 mM NA-Star substrate was added to each well and incubated at 34uC for 30 min. The luminescence signals for triplicate samples were counted for 1.0 second with a 2.0 second delay after the injection of 60 ml of NA-Star accelerator using a LB941 Multimode Reader TriStar equipped with automatic injectors for the accelerator (Berthold Technologies GmbH & Co. KG., Bad Wildbad, Germany). Unit activity of sample neuraminidase was calculated using a standard curve obtained by a characterized neuraminidase, a highly purified neuraminidase from Arthrobacter ureafaciens (Nacalai Tesque, Kyoto, Japan) of known unit activity, where one unit is defined as the activity to release 1 mmol of N-acetylneuraminic acid from substrate of NAN-Lactose per min. Neuraminidase activity was expressed as the arbitrary units of luminescence signals or as calculated units.To prepare culture supernatants, these bacteria were incubated in Trypticase soy broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) overnight at 35uC under aerobic conditions, and the culture supernatants were clarified by centrifugation for 15 min at 1,7506g and filtrated by a syringe filter with a 0.45 mm Supor membrane (PALL, Port Washington, NY, USA) to exclude bacteria.
Neuraminidase Inhibition Assay with Zanamivir
Samples were diluted with PBS to give approximate neuraminidase activity of 5,000 arbitrary units of luminescence signals and mixed with an equal volume of ten-fold serial dilutions of zanamivir (from 10 mM to 0.1 nM in PBS) or DANA (from 10 mM to 0.1 mM in PBS). The final concentrations in the mixtures ranged from 5 mM to 0.05 nM for zanamivir and 5 mM to 0.05 mM for DANA. The mixtures were incubated at 37uC for 30 min and assayed for neuraminidase activity. The IC50 value was read on the inactivation dose response curve.
Plaque Assay
The infectivity of viruses was determined by plaque assay as described below. Ten-fold serial dilutions of virus samples were made in Hanks balanced salt solution (HBSS; GIBCO, Gland Island, NY, USA) and 0.1 ml of the dilutions was inoculated on MDCK cell monolayers in 6-well tissue culture plates (Corning, Lowell, MA, USA). After adsorption of the virus onto the cells for 30 min at room temperature, 1.6 ml of Leibovitz’s L15 medium (GIBCO, Gland Island, NY, USA) containing 0.6% SeaKem ME agarose (Lonza, Basel, Switzerland), 1.5% gelatin (Nacalai, Kyoto, Japan) and 2.5 mg/ml TPCK-trypsin was added to each well and allowed to solidify. The plates were incubated for 3 days at 34uC and the number of plaques was counted. Infectivity was expressed as plaque forming units (pfu) per ml.
Hemagglutination Assay
Two-fold serial dilutions of virus samples were made in 50 ml of phosphate buffered saline (PBS; GIBCO, Gland Island, NY, USA) in 96-well U-bottom plates (BD, Franklin Lakes, NJ, USA). To each well, 50 ml of 0.5% (v/v) chicken red blood cells (Nippon Biotest Laboratories, Tokyo, Japan) in PBS was added. The plates were kept at 4uC for 1 h, after which, the hemagglutination patterns were read and hemagglutination (HA) titers were determined from the last dilutions showing complete hemagglutination. One HA unit (HAU) was defined as the quantity of virus contained in 1 ml of virus suspension of HA titer 1.
Hemagglutination Inhibition Assay
Two-fold serial dilutions of saliva samples were made in 25 ml of PBS on 96-well U-bottom plates. Then 25 ml of indicator viruses (8 HAU/ml PBS) were added for each dilution, and the plates were incubated for 1 h at 37uC. To each well, 50 ml of 0.5% (v/v) chicken red blood cells in PBS were added. The plates were kept at 4uC for 1 h and then the hemagglutination pattern was read. The reciprocal of the highest dilution that completely inhibited hemagglutination was taken as the hemagglutination inhibition (HI) titer.
Virus Growth Inhibition Assay
The inhibition of virus growth by neuraminidase inhibitors in the presence or absence of bacterial neuraminidase was assayed by measuring the released progeny virus in the culture fluid of infected cells. MDCK cells seeded on 12-well tissue culture plates were inoculated with 50 ml of virus suspension at a MOI of 0.001 or 0.01. After the adsorption for 1 h on ice, 1 ml MEM supplemented with 2.5 mg/ml TPCK-trypsin containing neuraminidase inhibitor and/or bacterial neuraminidase was added to each well. At the indicated times of incubation at 37uC in 5%CO2, culture media were harvested and the virus titers, performed in triplicate wells, were determined by plaque assay.
Infectivity Neutralization Assay
The infectivity-neutralization activity of saliva samples were assessed by measuring plaque reduction. Ten-fold serial dilutions of saliva samples were mixed with an equal volume of virus suspension (approximately 10,000 pfu/ml) containing 500 nM zanamivir with or without Vibrio cholerae RDE (460 munits/ml neuraminidase activity) and incubated for 1 h at 37uC. The mixtures were then titrated for infectivity by plaque assay. To avoid effects of zanamivir and RDE on plaque formation, cells were washed with 1.6 ml of HBSS after virus adsorption in the plaque assay. The dilution to give 50% reduction of pfu was estimated using the plaque-reduction curve and taken as the infectivity neutralization titer.30 min with rabbit polyclonal antibody against purified A/ Udorn/72 virions [42] in PBS containing 1% bovine albumin (fraction V, Reheis Chemical Company, Phoenix, Arizona, USA). After two washes, the cells were incubated for 30 min with Alexa Fluor 647 goat anti-rabbit IgG (Life Technologies Japan, Tokyo, Japan) in PBS containing 1% bovine albumin and Hoechst 33342 (to counterstain nuclei of all cells). The coverslips were mounted on a slide glass with ProLong Gold antifade reagent (Life Technologies Japan, Tokyo, Japan) and the cells were observed using a fluorescence microscope (BZ-8000, Keyence, Osaka, Japan). Acquired images were analyzed by using the software attached to BZ-8000.