Development of real‐time cell analysis methods applied to equine influenza virus: proof of concept

International audienceBackground: Equine influenza virus (EIV) is a respiratory pathogen that causes important economic losses to the equine industry. As a result, equine influenza has benefited from several technological advances in the fields of vaccine development, diagnosis and epidemiological surveillance. New Real-Time Cell Analysis (RTCA) methods (e.g. impedancemetry) allows sensitive measurement of EIV infection, tropisms and replication in vitro. This technological approach has now been applied to several equine viruses (e.g. equine herpesvirus; West Nile Virus) but has not been adapted to EIV yet. Objectives: To develop a RTCA model for EIV. Study design: In vitro experiments, proof of concept. Methods: 1) Real-time EIV SeroNeutralisation assay (RSNA) was applied to equine serums (n = 5) with different Single Radial Haemolysis (SRH) antibody titres and 2) antiviral compounds activity against EIV were screened. Both assays used MDCK cells. The normalised Cell Index (CIn) was calculated after 30 minutes pre-incubation of EIV A/equine/Jouars/4/2006 (H3N8, Florida Clade 2 sub-lineage) strain with different equine serums and subsequent cell infection. SRH titres ranged from 0mm² to 252mm². Antiviral compounds Zanamivir and Memantine were used at concentrations ranging from 50 to 1.56 µg/mL. Experimental CIn were compared with control conditions (i.e. cell-culture with/without EIV). Results: The CIn decrease induced by EIV infection was significantly reduced (p < 0.05) after pre-incubation with the reference EDQM serum (200mm²) and the high SRH titre serums (222 to 253 mm²). Serums with an intermediate or negative SRH titre (123 and 0 mm², respectively) did not prevent the CIn decrease induced by EIV. Zanamivir was significantly active against EIV when used at 12.5 µg/ml (p < 0.05). Memantine was not active against EIV at the concentrations used. Main limitations: Limited number of serums and EIV strains tested. Conclusions: RTCA could be used to develop new EIV neutralisation assays and to facilitate the screening of new antiviral molecules