Yellowfin seabream (Acanthopagrus latus) is one of the most commercially important marine fish in China. In this study, a new continuous cell line, named ALS cells, was developed from the spleen tissue of A. latus. The cell line was maintained in Dulbecco\'s modified Eagle medium/Nutrient Mixture F-12 Ham (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and successfully cultured up to 50 passages. The cell line was authenticated by amplifying and sequencing mitochondrial cytochrome C oxidase subunit-I (coi-I) gene. The ALS cell line had the maximum growth rate in DMEM/F-12 medium containing 20% FBS at 27°C. Chromosome number analysis showed that the ALS cells have a modal diploid chromosome number of 34. The ALS cell line was transfected with the pEGFP-N1 plasmid, and green fluorescence was observed. The ALS cell line was used for testing Mandarinfish ranavirus (MRV) susceptibility, and the cytopathic effects in the cell line were observed at 4 days post-infection (dpi). Furthermore, the susceptibility of the ALS cell line to MRV and the levels of MRV mRNA and viral loads were found to be significantly increased at 1-7 dpi. This study revealed that the ALS cell line could be useful for molecular, virological, and biotechnological studies on yellowfin seabream.

Mandarinfish ranavirus (MRV), also known as a variant of largemouth bass virus (LMBV), is an emerging pathogen in mandarinfish aquaculture. In this study, monoclonal antibodies (mAbs) against MRV were produced and characterized, and 7 mAbs were obtained through Western blotting screening and all 7 mAbs specifically recognized MRV/LMBV but not several piscine iridoviruses as ISKNV, GIV and TFV. By LC MS/MS analysis, the recognized viral proteins by seven mAbs were identified as MRV-pORF47L, MRV-pORF55R, MRV-pORF57L, MRV-pORF77L and MRV-pORF78L, respectively, and all five viral proteins are late expression structural proteins by Western blotting. Based on mAb 1C4, immuno-histochemistry and immuno-histo-fluorescence were performed to re-assess the tissue tropism of MRV. The result showed that abundant reactive signals were observed in infected spleen, kidney as well as intestine and pyloric caecum. Real-time quantitative PCR also demonstrated that spleen as well as pyloric caecum and intestines are the major target tissue upon MRV infection. In infected intestines and pyloric caecum, numerous enlarged, multinucleated cells with intracytoplasmic inclusions were identified as the target cells of MRV, suggesting that MRV serves as a digestive tract pathogen to mandarinfish, which may explain why acute infection of MRV can cause the typical clinicopathology featured by severe ascites.