Establishment of an immortalized renal epithelial cell line derived from grass carp and its susceptibility to grass carp Reovirus genotype II.

Hemorrhagic disease in grass carp (Ctenopharyngodon idella) caused by grass carp reovirus genotype II (GCRV-II) threatens sustainable aquaculture. To facilitate virus isolation, characterization, and studies on the pathogenic mechanisms of GCRV-II, we established a new cell line, Ctenopharyngodon idella trunk kidney (CITK). This cell line was developed using a trypsin digestion method and exhibited optimal proliferation at 27 °C in Medium 199/Leibovitz's L-15 medium supplemented with 10 % fetal bovine serum (FBS). Karyotype analysis revealed chromosome numbers of 2n = 48 and 2n = 60 at passages 10 (P10) and 60 (P60), respectively, indicating that the cell line underwent spontaneous immortalization during serial passaging. Species authentication (the mitochondrial cytochrome oxidase subunit I gene (cox1)), along with tissue-specific marker analysis, confirmed that CITK originated from the kidney tissue of grass carp. Mycoplasma testing confirmed the absence of contamination during cultivation. Functional assays demonstrated that CITK cells support efficient transfection with the green fluorescent protein gene plasmid. Notably, CITK exhibited significantly higher susceptibility to GCRV-II HN2407 compared to four other grass carp cell lines, with viral load reaching 6.26 × 105 copies/μL as determined by qRT-PCR. Kinetic analysis of infection revealed that, at 12, 24, and 48 h post-infection, GCRV-II induced time-dependent expression of key genes involved in innate immunity, pattern recognition receptors, signaling transducers, and adaptive immunity. The establishment of the immortalized CITK cell line provides a valuable platform for elucidating the pathogenic mechanisms of GCRV-II, developing antiviral strategies, and advancing studies on host-virus interactions in grass carp.