The reverse genetics applied to fish RNA viruses

Abstract

Aquaculture has expanded rapidly to become a major economic and food-producing sector worldwide these last 30 years. In parallel, viral diseases have emerged and rapidly spread from farm to farm causing enormous economic losses. The most problematic viruses encountered in the field are mainly, but not exclusively, RNA viruses belonging to the Novirhabdovirus, Aquabirnavirus, Alphavirus and Betanodavirus genera. The recent establishment of reverse genetics systems to recover infectious fish RNA viruses entirely from cDNA has made possible to genetically manipulate the viral genome. These systems have provided powerful tools to study all aspects of the virus biology and virus-host interactions but also gave the opportunity to use these viruses as live vaccines or as gene vectors. This review provides an overview on the recent breakthroughs achieved by using these reverse genetics systems in terms of viral protein function, virulence and host-specificity factor, vaccine development and vector design.

Figure 1

Systems of reverse genetics for fish RNA virus generation entirely from cloned cDNA. Methods used for the recovery from plasmid cDNA of fish RNA viruses, belonging to the Aquabirnavirus, Novirhabdovirus, Betanodavirus and Alphavirus genera, are presented. A schematic representation of each plasmid cDNA encoding viral replicative complex proteins or containing a cDNA copy of full-length viral genome, antigenome or genome segments is drawn. Plasmid DNA or in vitro transcribed RNA were transfected (red arrows) in either untreated fish permissive cells (CHSE-214, E-11 and BF-2 cells) or permissive cells constitutively expressing the T7RNAP (CELLS-T7: fish EPC-T7 cells or Baby Hamster Kidney derived BSR-T7/5 cells) or previously infected with a recombinant vaccinia virus (vTF7-3) encoding the T7RNAP (EPC and BF-2 cells). After the initiation of an infectious cycle, recombinant virus can be harvested (blue arrows). For further details, see section 2. Abbreviations are: T7p, T7 promoter; CMV, cytomegalovirus promoter; HH, hammerhead ribozyme sequence; NC, non-coding region; Sgt, viral genome segment; pA, polyadenylation signal; δ, antigenomic sequence of a ribozyme derived from the hepatitis delta virus; T7t, T7 terminator; ©, cap structure; T7RNAP, T7 RNA polymerase.