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Vibrios belonging to the Harveyi clade are pathogenic marine bacteria affecting both vertebrates and invertebrates, thereby causing a severe threat to the aquaculture industry. In this study, the expression of haemolysin, metalloprotease, serine protease, the quorum sensing master regulator LuxR and the virulence regulator ToxR in different Harveyi clade isolates was measured with reverse transcriptase real-time PCR with specific primers. There was relatively low variation in thein vitroexpression levels of the quorum sensing master regulatorluxR(sevenfold), whereas for the other genes, the difference in expression between the isolates showing lowest and highest expression levels was over 25-fold. Furthermore, there was a significant correlation between expression levels oftoxRandluxRand between the expression levels of these regulators and the protease genes. The expression levels ofluxR,toxRand haemolysin were negatively correlated with the survival of brine shrimp larvae challenged with the isolates. Finally, a non-virulent, a moderately virulent and a strongly virulent isolate were selected to studyin vivoexpression of the virulence genes during infection of gnotobiotic brine shrimp larvae. Thein vivogene expression study showed a clear difference in virulence gene expression between both virulent isolates and the non-virulent isolate.