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A new marine fish cell line, EAGL, derived from the liver of red-spotted grouper Epinephelus akaara was established and characterized. The cells multiplied well in minimum essential medium (MEM) supplemented with 10% foetal bovine serum (FBS) at temperatures between 25 and 30° C. The growth rate of this cell line increased as the proportion of FBS increased from 5 to 20% at 25° C, with maximum growth at the concentration of 15 or 20% FBS. Morphologically, the cells were epithelial-like and the presence of pancytokeratin confirmed their epithelial origin. Chromosome analysis revealed that the modal chromosome number was 48. The susceptibility of the cell line to four fish viruses was tested. Significant cytopathic effect (CPE) was only observed in Singapore grouper iridovirus (SGIV)-infected cells, and the virus replication was further confirmed by immunofluorescence, electron microscopy and real-time reverse-transcription (RT)-PCR assay. When the cells were transfected with pEGFP-N3 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies.

BackgroundshRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV) receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT™, which expressed siRNA targeting the FMDV receptor, the porcine integrin αv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF) and cell enzyme linked immunosorbent assays (cell ELISA), and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells.ResultsOur results indicated successful establishment of the iαv U6 RNAi entry vector and the iαv pLenti6/BLOCK -iT expression vector. The functional titer of obtained virus was 1.0 × 106 TU/mL. To compare with the control and mock group, the iαv-PK-15 group αv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold and there was a > 99% reduction in virus titer when cells were challenged with 102 TCID50 of FMDV.ConclusionsIαv-PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.