Fish gills are anatomically complex tissues that perform a multitude of functions and often comes into direct contact with variable environmental parameters such as salinity, random ion compositions, and pH fluctuations (Hwang and Lee, 2007). They are responsible for osmoregulation, ion regulation and ammonia excretion which are crucial for the overall well-being of the fish species. Owing to their direct exposure to the environment, gills are vulnerable to contaminants, toxins, and pathogenic microbes, which can easily disrupt the basic functions and alter tissue homeostasis (Zawisza et al., 2024). Previous studies suggest that gills act as the port of entry for bacteria (Campos-perez et al., 2000, Ling et al., 2001, Løvoll et al., 2009, Tobback et al., 2009), viruses such as infectious hematopoietic necrosis virus (IHNV) and infectious salmon anaemia virus (ISAV) (Drolet et al., 1994, Totland et al., 1996), and parasites (Belem and Pote, 2001) from the aquatic environment.

Fish cell lines are used to create suitable study models for toxicological, pathological and immunological studies (Goswami et al., 2022). In vitro approaches using cell lines can serve both diagnostic and research purposes. Examples from previous studies include the utilization of a trout cell line used to screen immune genes during an infection by the fish pathogenic oomycete Saprolegnia parasitica, and establishment of a fish gill cell line to investigate protozoan parasite infections in yellow sea bream (De Bruijn et al., 2012, Li et al., 2024). The initial establishment of a primary culture of gill epithelium was achieved using the single-seeded insert technique (SSI). This technique involved a two-chamber system with a permeable barrier serving as a separator, which facilitated the growth of cells into polarized epithelium (Pärt et al., 1993; Wood and Pärt, 1997). This setup comprised an apical (water-filled) and basolateral (blood/media) compartment, designed to mimic properties similar to the gill epithelium in fishes. To overcome the limitation of a single cell population forming the epithelial layer, advancements were made using the double-seeded insert (DSI) technique. This technique facilitated the formation of a tight epithelial gill cell layer composed of multiple cell types such as pavement cells and mitochondrion-rich cells. Such a configuration allowed for enhanced ion movement between compartments and efficient Ca2+ transport, similar to the functioning of fish gills in live organisms (Fletcher et al., 2000, Kelly and Wood, 2008, Walker et al., 2007, Zhou et al., 2003). Primary gill cell culture offers advantages over cell lines owing to the presence of various cell types that better mimic gill physiology and are more suitable for functional studies (Bols et al., 1994).

Nervous necrosis virus (NNV) afflicts over 120 species in aquatic environments, posing a significant economic burden on aquaculture (Doan et al., 2017). This neurotrophic virus selectively targets neuronal tissues, whereas gills, being non-neuronal tissues, serve as entry points where the virus replicates and proliferates before infiltrating the central nervous system of the fish (Lampou et al., 2020, Rombout et al., 2014). Given the substantial impact of NNV on aquaculture, numerous studies are underway to comprehend its host pathogen interactions. Krishnan et al.,(2020), conducted studies on NNV infection in the sevenband grouper, concluding that gills serve as the primary entry point for the pathogen. Consequently, in this study, a 2D cell culture system utilizing primary gill cells from the sevenband grouper (Hyporthodus septemfasciatus) was developed as a technique to validate and closely monitor host-pathogen-specific interactions.