Decomposition of L-glutamine and accumulation of ammonium in cell culture media inhibit infectivity of influenza viruses

Cationic lysosomotropic molecules such as ammonium salts and chloroquine inhibit influenza A virus (IAV) infection in cell culture by counteracting endosomal acidification and hampering viral-endosomal fusion. Here, we studied the effects of storage of L-glutamine-supplemented cell culture media on accumulation of ammonium and inhibition of IAV infection. The storage-related inhibitory effect was observed for DMEM and OptiMEM media but not for RPMI medium, and was more pronounced for IAV with pH-stable hemagglutinin. Our results highlight the importance to consider potential presence of virus-inhibiting lysosomotropic agents and/or their production in culture media with negative effect on influenza virus infection. •Improper cell culture medium handling releases ammonium via decomposition of L-glutamine.•Using old or pre-warmed cell culture medium reduces infectivity of influenza A viruses with stable hemagglutinin.•The buffer substance HEPES preserves influenza A virus infectivity.