Emerging fish diseases can cause significant economic losses for aquaculture producers. Cyprinid herpesvirus-3 (CyHV-3), also known as Koi Herpesvirus (KHV), an enveloped double-stranded DNA virus, is the aetiological agent of the highly contagious koi herpesvirus disease (KHVD) of wild and cultured carp (Cyprinus carpio). RNA interference (RNAi) is a natural inhibitory mechanism of gene expression. It was first demonstrated by Fire et al. who injected double stranded RNA (dsRNA) into the worm Caenorhabditis elegans. The RNAi mechanism is initiated by dicer which cuts long dsRNA into small fragments between 21 and 25 nucleotides long. These are called small interfering RNA (siRNA). The RNAi mechanism can be found in invertebrates, plants and mammals including humans. RNAi could possibly be used as a method to control emerging viral infections where no other effective control methods exist. The idea that RNAi could be used for viral inhibition is based on previous studies in plants where the process of viral replication was a target of this silencing mechanism. More recently, RNAi has been used in vitro to effectively suppress the replication of a number of fish viruses. In these cases, siRNAs are produced from short hairpin RNA (shRNA) expression plasmids that are transfected into the appropriate cell line prior to infection with virus. In order to investigate the efficacy of RNAi in the suppression of CyHV-3 in vitro in susceptible KF-1 cells, firstly the optimal conditions for transfection had to be determined. Transfection of shRNA expression plasmids into KF-1 cells by means of a chemical (Fugene HD) had low toxicity for the cells but the efficacy of transfection was low. On the other hand, transfection by electroporation had greater efficacy (although only with program X-01 from the Amaxa nucleofector), and it resulted in high viability of cells. shRNAs are one source of specific siRNAs to control particular viruses. To support expression of shRNA in KF-1 cells, U6 promoters from zebrafish, mouse and fugu were tested. The zebrafish U6-3 promoter, which had previously been tested for activity in ZF4 cells was also found to be the best for expressing pEshRNAGFP in KF-1 cells. By contrast, fugu and mouse U6 promoters did not permit expression of pEshRNAGFP in KF-1 cells. Potential target genes of CyHV-3 for RNAi inhibition in vitro were selected and validated. Two IE (ORF1 and ORF3) and two E genes (DNA polymerase, DNP, and major capsid protein, MCP) were chosen and two shRNA plasmids were constructed for each target. Four of eight shRNAs, DNP-3, MCP-4, ORF3-13 and ORF3-14, delayed viral replication at 2 dpi in an initial experiment. In a repeat experiment with duplicate samples, shRNA ORF3-14, targeting an IE gene, resulted in an approximately 10 fold inhibition of viral replication and viral genome copy number from 6-48 hpi. shRNA ORF3-13 caused more limited inhibition between only 24-36 hpi. shRNA MCP-4, targeting an E gene, also showed some inhibition of CyHV-3 from 6-48 hpi, either causing an approximately 10 fold reduction in virus replication for a short period, or a reduction in viral genome copy number for a longer period. These data provide evidence for potential CyHV-3 gene targets, either alone or in combination, if RNAi were to be considered as a future form of control of KHVD in farmed carp.