Development of immortalized feline respiratory epithelial cells in an air-liquid-interface culture system for feline herpesvirus-1 study

Feline herpesvirus-1 (FHV-1) is responsible for approximately 50% of diagnosed viral upper respiratory tract disease in cats. The virus infects and replicates in the epithelial cells located in upper respiratory tract. Com-mercial vaccines do not protect cats from the infection itself or development of latency. Previously, our lab developed a cell culture model using primary feline respiratory epithelial cells (pFRECs) to study respiratory innate immunity to FHV-1 and FHV-1 deletion mutants. However, the numbers of pFRECs that can be obtained per cat is limited. To improve the usage of respiratory epithelial 3D cultures in FHV-1 research, the present study immortalized feline respiratory epithelial cells (iFRECs) and characterized them morphologically and immuno-logically and evaluated the response to FHV-1 infection. Immortalization was achieved by transduction with Lenti-SV40T and Lenti-HPV E6/E7. Immortalized FRECs could be successfully subcultured for >20 passages, with positive gene expression of SV40T and HPV E6/E7. Immortalized FRECs expressed similar innate immunity -associated genes compared to pFRECs, including genes of Toll-like receptors (TLR1-9), interferon induced genes (OAS1, OAS3, IFI44, IFITM1, IFIT1), chemokines (CCL2, CCL3, CXCL8), pro-inflammatory and regulatory cy-tokines (IL-6, IL-4, IL-5, IL-12, and IL-18), and antimicrobials (DEF010, DEF04B). Finally, FHV-1 inoculation resulted in characteristic cytopathic effects starting at 24 hpi, with more than 80% cells detached and lysed by 72 hpi. Overall FHV-1 growth kinetics in iFRECs resembled the kinetics observed in pFRECs. In conclusion, we demonstrated that iFRECs are a useful tool to study feline respiratory disease including but not limited to FHV-1.