Abstract
The genome sequence of a hypervirulent novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) French strain 23-75, was determined. Compared to the genome of the prototype Fil3 strain, a number of substitutions, deletions, and insertions were observed. Following the establishment of a plasmid-based minigenome replication assay, recombinant VHSV (rVHSV) was successfully recovered. rVHSV exhibits wild-type-like growth properties in vitro as well as in vivo in rainbow trout. The dispensable role of NV for the novirhabdovirus replication was confirmed by generating rVHSV-DeltaNV, in which the NV gene was deleted. This deletion mutant was shown to be as debilitated as that previously described for infectious hematopoietic necrosis virus (IHNV), a distantly related novirhabdovirus (S. Biacchesi, M. I. Thoulouze, M. Bearzotti, Y. X. Yu, and M. Bremont, J. Virol. 74:11247-11253, 2000). Recombinant VHSV and IHNV expressing tdTomato and GFP(max) reporter genes, respectively, were generated, demonstrating the potential of these rhabdoviruses to serve as viral vectors. Interestingly, rIHNV-GFP(max) could be recovered using the replicative complex proteins of either virus, whereas rVHSV-Tomato could be recovered only by using its own replicative complex, reflecting that the genome signal sequences of VHSV are relatively distant from those of IHNV and do not allow their cross-recognition. Moreover, the use of heterologous protein combinations underlined the importance of strong protein-protein interactions for the formation of a functional ribonucleoprotein complex. The rIHNV-GFP(max) and rVHSV-Tomato viruses were used to simultaneously coinfect cell monolayers. It was observed that up to 74% of the cell monolayer was coinfected by both viruses, demonstrating that a limited interference phenomenon exists during the early stage of primary infection, and it was not mediated by a cellular antiviral protein or by some of the viral proteins.