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Background  Envelope protein 53R was identified from frog  Rana grylio  virus (RGV), a member of the family  Iridoviridae , and it plays an important role in the virus assembly. Although inhibition of iridovirus major capsid protein (MCP) by small hairpin RNAs (shRNAs) has been shown to cause resistance to viral infection  in vitro , RNA interference (RNAi) to inhibit aquatic animal virus envelope protein gene product has not been reported.    Methodology  We devised artificial microRNAs (amiRNAs) that target a viral envelope protein gene RGV  53R . By incorporating sequences encoding amiRNAs specific to  53R  of RGV into pre-miRNA155 (pSM155) vectors, which use the backbone of natural miR-155 sequence and could intracellularly express  53R -targeted pre-amiRNAs. The pre-amiRNAs could be processed by the RNase III-like enzyme Dicer into 21–25 nt amiRNAs (amiR-53Rs) in fish cell lines. The levels of  53R  expression were analyzed through real-time PCR and RGV virions assembly were observed by electronic microscopy in fish cells transfected with or without amiR-53Rs at 72 h of RGV infection.    Conclusion/Significance  The results argue that viral envelope protein RGV  53R  can be silenced and the virions assembly was deficient  by a miR-53R-1, and further identified the first amiRNA of envelope protein gene from iridovirus that was able to cause resistance to virus infection in fish cells. The data demonstrate that the viral infection is efficiently suppressed (58%) by amiR-53R-1 targeting positon 36–57 of RGV  53R . Moreover, electron microscopic observations revealed virion assembly defect or reduced virions assembly capacity was closely correlated to expression of amiR-53R-1. Based on real time PCR of the  Mx  gene, we found no evidence of activation of IFN by amiR-53R-1.

Viral Envelope Protein 53R Gene Highly Specific Silencing and Iridovirus Resistance in Fish Cells by AmiRNA