Arthrobacter ureafaciens neuraminidase

Hemagglutination (HA) assay was performed with giant panda, human, rat, pig and rabbit erythrocytes (0.5% suspension in PBS). Two-fold serial dilutions of viruses, starting at 10⁴ TCID₅₀ per well, were mixed 1:1 with the erythrocytes in a V-shape plate (Corning™, 3894). Hemagglutination was assessed after 2-hour incubation on ice, and hemagglutinating units (HAU) were inspected for erythrocytes of each species. For desiaylation, erythrocytes were treated with Arthrobacter ureafaciens neuraminidase (Roche, 10mU/mL) for 3 hours at 37°C, and washed 5 times with cold PBS prior to the HA assay.
Hemagglutination inhibition (HI) assays were carried out to study the HA inhibition ability of different serum samples. Different samples were two-fold serially diluted, and mixed 1:1 with PBS containing 8 HAU of pFPV-sc. The mixtures were incubated at room temperature for one hour, and then mixed 1:1 with human erythrocytes (0.5% in PBS). Hemagglutination inhibition was documented after 2-hour incubation on ice. The HI titer of one serum sample is conveyed as the reciprocal of the highest serum dilution that still shows hemagglutination inhibition.

Protein/spike histochemistry was performed as described previously [11 (link)] (schematically depicted in Fig 2C). TMA sections were rehydrated, treated with 1% hydrogen peroxide to remove endogenous peroxidases and goat serum to block nonspecific binding. Next, precomplexed S1/HA proteins (20–30 μg of S1 and 2–3 μg of HA per TMA) and Strep-Tactin HRP were applied to TMAs and incubated at 4°C overnight. AEC (3-amino-9-ethylcarbazole, Dako, The Netherlands) substrate was used to detect binding of proteins. To remove sialic acids, tissues were treated with Arthrobacter ureafaciens neuraminidase (Roche, USA) at a concentration of 1 mU/100 μl in PBS (pH 5.0) overnight at 37°Ci

Cell suspensions of SK-N-SH or RD cells were incubated with 100 mU/ml Arthrobacter ureafaciens neuraminidase (Roche) or 10 mIU/ml heparinase III (Sigma-Aldrich) in serum-free medium for 1 h at 37°C. To prevent the cell surface sulfation, cells were cultured for 5 days with 80 mM sodium chlorate (NaClO₃; Sigma-Aldrich, 1.06420 EMD Millipore). Removal of α(2,3)-linked sialic acid and α(2,6)-linked sialic acid on the cell surface was confirmed by staining with fluorescein-labeled Sambucus nigra lectin (SNA) (5 μg/ml; Vector Laboratories, CA, USA) and biotinylated Maackia amurensis lectin (MAL) I (5 μg/ml; Vector Laboratories). The sialylated cells were detected by streptavidin-conjugated Alexa Fluor 488 (5 μg/ml; Thermo Fisher Scientific). Removal of heparan sulfate on the cell surface was confirmed using a mouse anti-heparan sulfate monoclonal antibody (10 μg/ml; Amsbio, Frankfurt, Germany). No-virus inoculation control and virus inoculation in nonenzymatic treatment control were included as a negative control and positive control, respectively, in all assays. Mean fluorescence intensity (MFI) was measured with BD FACS Lyrics (BD Bioscience). Subsequently, virus attachment was determined as described above. Data were analyzed using FlowJo 10 software (Ashland, OR, USA). The experiments were performed at least 3 times, and each experiment was performed in duplicate.