Single-cell investigation of the influenza A virus neurotropism in vitro.

Despite influenza A viruses (IAVs) are notorious respiratory pathogens, some variants behave neurotropically and are therefore able to reach and replicate in the central nervous system. Especially high-pathogenic avian IAVs such as H7N7 and H5N1 pose a risk for future pandemics and exert a broad neurotropic potential linked to the pathogenesis of neurodegenerative diseases in vivo. There is a lack of understanding in the viral and cellular reactions once the virus reaches and encounters cells within the brain parenchyma. Here, a primary murine triple co-culture system consisting of neurons, astrocytes and microglia was infected with neurotropic H7N7 and non-neurotropic H3N2 to understand the IAV neurotropism in vitro. An high-pathogenic avian strain of subtype H5N1 was also included to investigate the zoonotic potential between the three IAV strains. A self-written analysis script in python was utilized for the image-based single-cell analysis. To analyze cellular reactions towards neurotropic IAV reactions a bead-based multiplex assay followed by analysis via flow cytometry was performed. Moreover, a live-cell assay was utilized investigating the caspase-3/7 activation in specific cell populations upon IAV infections. Last, the ability of different herbal teas and their compounds was tested to inhibit IAV infections in the lung. A mechanism is suggested in which microglia, brain-resident macrophages, provide an initial line of defense against IAVs. The activation of caspase-3/7 appear to decide between microglial activation and cell death, with the latter aborting the viral replication. These signals might further induce astrocytes, at higher viral loads, to step in as a secondary barrier to restrict the IAV replication and protect neurons. Astrocytes, however, appeared to act as the main virus producers and promoted the viral spreading, especially at higher virus concentrations. In contrast, the cells in the triple co-culture were able to restrict the propagation of non-neurotropic H3N2 infection at least at lower virus concentrations. This mechanism was accompanied by an upregulation of early immune cues such as Monocyte-chemoattractant protein 1 (MCP-1). Last, herbal tea compounds exerted antiviral effects on the IAV infection in lung cells providing a therapeutic approach to treat Influenza. Future research is necessary to validate these findings. Together, this provides a mechanistic explanation about cellular and viral reactions in the initial phase of IAV infections in neuronal cells.