Immunofluorescence‐Mediated Detection of Respiratory Virus Infections in Human Airway Epithelial Cultures

Abstract

A diverse collection of viral pathogens target airway epithelial cells for infection, with effects ranging from mild upper respiratory tract symptoms to death of the infected individual. Among these pathogens are recently discovered and/or emergent viruses that sometimes fail to infect commonly used, immortalized cell lines and for which infection phenotypes in the respiratory tract remain unknown. Human airway epithelial cultures have been developed over the past several decades and have proven to be a useful model system in culturing hard-to-grow viruses and assaying various features of infection in a physiologically relevant setting. This article includes methods for the generation of well-differentiated human airway epithelial cell cultures at air-liquid interface that recapitulate the mucosal epithelium of the trachea/bronchus in vivo. We further detail inoculation of these cultures with respiratory viruses-specifically rhinovirus, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-and provide a protocol for the detection of double-stranded RNA or viral antigen-positive cells by immunofluorescence microscopy. These techniques, together with a post-imaging analysis, can be applied to characterize the efficiency of infection and kinetics of spread within the airway epithelium. Furthermore, these methods can be utilized in conjunction with antibodies against cellular targets to determine cell tropism and colocalization with specific host factors during infection.

Live imaging of the airway epithelium reveals that mucociliary clearance modulates SARS-CoV-2 spread

“…RSV and human parain uenza virus may display comet shaped foci early in infection, but for these viruses the foci merge and grow to occupy the majority of the culture surface area, rather than remaining distinct as in SARS-CoV-2 in the current study 5,30 . For in uenza A virus, while comet shaped foci have been anecdotally reported (personal communication), it appears to be a relatively uncommon phenomenon and no published images were identi ed 33,34 . Comparisons between viruses based on published literature are suspect owing to differences in infection, culture conditions, and imaging protocols.…”

Progress in etiological diagnosis of viral meningitis

“…It has been shown that measurement of calcitonin using antibody-conjugated fluorospheres can differentiate atypical bacterial meningitis from viral encephalitis in children ( 49 ). Immunofluorescence microscopy enables single-cell analysis of samples and preserves spatial information within cells as well as throughout the culture, and can be paired with image analysis tools to localize to the virus or detect expression of associated viruses at the single-cell level, but this technique is only useful in the absence of infected cells ( 50 ).…”