Journal: bioRxiv

Article Title: Impact of pH and temperature in dairy processing on the infectivity of H5N1 avian influenza viruses

doi: 10.1101/2025.03.21.644501

Figure Lengend Snippet: ( a ) Determination of the pH threshold triggering HA-mediated membrane fusion. Vero cells were infected with VSVΔG(HA:NA:GFP) encoding the HA and NA glycoproteins of HPAIV A/Pelican/Bern/1/2022 (H5N1) along with green fluorescent protein (GFP). At 18 hours post infection, the cells were exposed for 10 minutes at 37 °C to buffers adjusted to the indicated pH values and subsequently incubated for 2 hours at 37°C in MEM medium. The cells were fixed and nuclei stained with DAPI. The cells were visualized by fluorescence microscopy (nuclei: blue fluorescence; GFP: green fluorescence). Bar size = 50 μm. Arrows point to syncytia formed. ( b-d ) Analysis of pH-dependent inactivation of H5 avian influenza viruses. HPAIV A/cattle/Texas/063224-24-1/2024 (H5N1) ( b ), LPAIV A/duck/Hokkaido/Vac-1/2004 (H5N1) (b), and LPAIV A/duck/Potsdam/1402/1986 (H5N2) ( c ) were incubated for 30 minutes at 21 °C with buffers adjusted to the indicated pH values. The buffers were subsequently replaced by MEM medium using Sephadex G25 size exclusion chromatography, and infectious virus titers determined on MDCK cells. Mean values and standard deviations of three inactivation experiments are shown. Significantly different titers compared to the treatment at pH 7.0 were calculated using the one-way ANOVA test with SIDAK’s multiple comparisons (** p < 0.01, **** p < 0.0001).

Article Snippet: To replace the buffer with cell culture medium, each incubation (500 μL) was loaded on a Sephadex G-25 columns (PD MiniTrap G-25, Cytiva Europe, Freiburg, Germany) that has been equilibrated with MEM medium containing 5% FBS and peniciliin/streptomycin (Life Technologies, Zug, Switzerland).

Techniques: Membrane, Infection, Incubation, Staining, Fluorescence, Microscopy, Size-exclusion Chromatography, Virus