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. Author manuscript; available in PMC: 2010 Dec 1.
Published in final edited form as: Antiviral Res. 2009 Sep 20;84(3):215–224. doi: 10.1016/j.antiviral.2009.09.005

Figure 2. Functional analysis of influenza VLPs.

Figure 2

(a). Hemagglutination activity of influenza VLPsSerial dilutions of influenza VLPs, inactivated influenza viruses, or control rift valley fever virus VLPs in 50 μl of PBS were added to each wells of a v-bottom plate, followed by addition of 50 μl chicken red blood cells (0.5%). Hemagglutination of chicken red blood cells was recorded after 45 min incubation at room temperature. VLP, influenza VLPs; Control, control rift valley fever virus VLPs; IIV, inactivated influenza virus. (b). Neuraminidase (NA) activity in influenza VLPs. Different amounts of influenza VLPs, control rift valley fever virus VLPs, or purified influenza viruses were incubated with a substrate solution (Amplex Red/HRP/galactose oxidase/fetuin) for 30 min at 37 °C. The levels of NA activities were determined by measuring the fluorescence activity at 530/590 nm (excitation/emission) wavelengths. Error bars represent standard deviations for each sample. VLP, influenza VLPs; Control, RVFV VLPs; Virus, purified influenza virus A/PR/8/34; Negative control, supplied with the assay kit. (c). Stimulation of mouse bone marrow-derived dendritic cells (BMDCs) by influenza VLPs. BMDCs were prepared and stimulated with purified influenza VLPs or IIV vaccines as described in Materials and Methods. DCs incubated with culture medium only or LPS (10 ng/ml) were used as negative and positive controls respectively. To ensure that stimulation of DC by VLPs or purified viruses was not due to contamination by endotoxin, the DCs were also incubated with the same amount of VLPs or IIV vaccines that had been heat-treated at 100 °C for 30 min. Cell-free supernatants were harvested 24 hr after incubation at 37 °C in 5% CO2 and assayed for the levels (pg/ml) of IL-6 and TNF-alpha by ELISA. Error bars represent standard deviations of the triplicate wells for each sample and the results shown represent typical results obtained in two different stimulation experiments. 1, Medium (negative control); 2, influenza VLPs (10 μg/ml); 3, IIV (10 μg/ml); 4, LPS (10 ng/ml, positive control).