Viral hemorrhagic septicemia virus (VHSV) is a member of the Novirhabdovirus genus in the Rhabdoviridae family. VHSV is considered by many countries and international organizations to be one of the most important viral pathogens of finfish (38). During recent years, VHSV has been isolated from at least 50 different species from marine and freshwater fish and is present throughout the northern hemisphere (45). The transmission of the virus from fish to fish occurs directly through the water or by contact between infected and healthy individuals. VHSV is thought to enter the body through the gills or possibly through wounds on the skin. However, we recently showed that fins may represent the main portal of entry for the novirhabdoviruses (25). The virus usually causes severe hemorrhages in the skin, muscles, eyes, kidney, and liver, with mortality rates as high as 90%. As for all members of the Rhabdoviridae family, the VHSV genome consists of a negative-sense single-stranded RNA molecule of about 11 kb encoding five structural proteins: N, the nucleoprotein; P, a polymerase-associated protein; M, the matrix protein; G, the unique viral surface glycoprotein; and L, the large RNA-dependent RNA polymerase. In addition, like the other members of the Novirhabdovirus genus, such as infectious hematopoietic necrosis virus (IHNV), the VHSV genome encodes a small nonstructural NV protein, which has been shown to be dispensable for IHNV replication in cell culture and is involved in virus-induced pathogenicity in rainbow trout (8, 50). The sequence analysis of the glycoprotein (G) and nucleoprotein (N) genes of VHSV has shown that VHSV isolates can be divided into four genotypes that generally correlate with geographic location rather than the host species (4, 19, 47, 49). Isolates belonging to VHSV genotypes I, II, and III are present in continental Europe, the north Atlantic Ocean, the Baltic Sea, the North Sea, and waters around Scotland. Genotype IV consists of isolates from the marine environment in North America. Recently, viral hemorrhagic septicemia has become an emerging disease of freshwater fish in the Great Lakes region of North America (2, 54). Thus, it is quite obvious that VHSV is becoming a worldwide and very-broad-host-range fish virus and that the development of efficient vaccines is needed. Reverse genetics, allowing the introduction of targeted modifications into the viral genome and the production of attenuated live vaccine, may help to fight this rapidly spreading and emerging virus. It is routinely observed in farm trouts exposed to viral diseases that VHSV and IHNV coexist (26). By developing experimental coinfections by VHSV and IHNV in rainbow trout, Brudeseth et al. studied the pathogenesis and virus distribution (10). They found that both viruses established an infection and raised similar virus titers in kidneys, but the distribution of IHNV was more restricted in internal organs during the acute stage of the infection and was not detected in the brain. However, it generally is admitted that infection by one virus renders host cells resistant to a superinfecting virus. Superinfection exclusion, also known as homologous interference, is the phenomenon in which a cell infected with one type of virus or transfected with a viral replicon becomes resistant to a secondary infection with the same virus, whereas infection with unrelated viruses normally is unaffected (40, 51). Superinfection exclusion has been observed in a broad range of viruses, including vaccinia virus (14, 18), human immunodeficiency virus (HIV) (36, 37), vesicular stomatitis virus (VSV) (32, 43, 53), Borna disease virus (BDV) (24), measles virus (34), Sindbis virus (28), Semliki Forest virus (44), rubella virus (15), hepatitis C virus (HCV) (40, 51), and bovine viral diarrhea virus (BVDV) (31). Mechanisms of exclusion are diverse and have not been determined in all cases, but mechanisms described so far are caused by competition among different viruses for critical replicative pathways (for example, the use of the same receptors for the entry) or depend on the direct interaction of products of the primary infection with the secondary infecting virus. For example, the superinfection exclusion of VSV was found to be caused by a combination of three distinct effects on endocytosis by VSV-infected cells: (i) a decreased rate of the formation of endocytic vesicles, (ii) a decreased rate of the internalization of receptor-bound ligands, and (iii) a competition with newly synthesized virus for the occupancy of coated pits (43). In contrast, the cytoplasmic accumulation of BDV nucleocapsid components appeared to prevent subsequent infection through a blockage of the polymerase activity of incoming viruses (24). Superinfection exclusion by BVDV was the result of dual mechanisms that were mediated by the structural protein E2, which blocks the entry of a homologous second virus, and by a blockage at the level of replication dependent on the level of primary viral RNA replication but not influenced by the expression of viral structural proteins, as observed for BDV (31). HIV employs its early gene product Nef to efficiently interfere with superinfection at the virus entry step by downregulating cell surface receptors (36). Finally, vaccinia virus expresses in newly infected cells two surface proteins that mark cells as infected and induce the repulsion of superinfecting viruses (18). In the present study, we described a reverse-genetics system for VHSV allowing the generation of a wild-type-like recombinant VHSV and a recombinant virus expressing a red fluorescent protein (Tomato). The system is based on the French strain 23/75 of VHSV, which is a hypervirulent and devastating strain for farmed rainbow trout belonging to genotype I (serotype III) and was isolated in France in 1975 from a brown trout (16, 23). Thus, this system provides a suitable starting point for identifying potential virulence determinants, as demonstrated by the deletion of the NV gene, and for developing attenuated derivatives as candidate vaccines. Using the available reverse-genetics system elaborated with IHNV, a recombinant IHNV expressing a green fluorescent protein (GFP) also was produced (8), and it was of interest to study whether a superinfection exclusion phenomenon could be observed between both VHSV and IHNV, whose cohabitation has been recorded often. We showed that up to 74% of a cell monolayer could be simultaneously infected by the viruses, demonstrating a limited viral interference between salmonid novirhabdoviruses and that, based on previous data, chimeric or pseudotyped viruses could be generated (6).