Although Novirhabdovirus viruses, like the Infectious hematopietic necrosis virus (IHNV), have been extensively studied, limited knowledge exists on the route of IHNV entry during natural infection. A recombinant IHNV (rIHNV) expressing the Renilla luciferase gene was generated and used to infect trout. A noninvasive bioluminescence assay was developed so that virus replication in live fish could be followed hours after infection. We provide here evidence that the fin bases are the portal of entry into the fish. Confirmation was brought by the use of a nonpathogenic rIHNV, which was shown to persist in fins for 3 weeks postinfection.

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.

Sleeping disease virus (SDV) is a member of the new Salmonid alphavirus genus within the Togaviridae family. The single-stranded RNA genome of SDV is 11,894 nucleotides long, excluding the 3′ poly(A) tail. A full-length cDNA has been generated; the cDNA was fused to a hammerhead ribozyme sequence at the 5′ end and inserted into a transcription plasmid (pcDNA3) backbone, yielding pSDV. By transfection of pSDV into fish cells, recombinant SDV (rSDV) was successfully recovered. Demonstration of the recovery of rSDV was provided by immunofluorescence assay on rSDV-infected cells and by the presence of a genetic tag, a BlpI restriction enzyme site, introduced into the rSDV RNA genome. SDV infectious cDNA was used for two kinds of experiments (i) to evaluate the impact of various targeted mutations in nsP2 on viral replication and (ii) to study the virulence of rSDV in trout. For the latter aspect, when juvenile trout were infected by immersion in a water bath with the wild-type virus-like rSDV, no deaths or signs of disease appeared in fish, although they were readily infected. In contrast, cumulative mortality reached 80% in fish infected with the wild-type SDV (wtSDV). When rSDV-infected fish were challenged with wtSDV 3 and 5 months postinfection, a long-lasting protection was demonstrated. Interestingly, a variant rSDV (rSDV ₁₄ ) adapted to grow at a higher temperature, 14°C instead of 10°C, was shown to become pathogenic for trout. Comparison of the nucleotide sequences of wtSDV, rSDV, and rSDV ₁₄ genomes evidenced several amino acid changes, and some changes may be linked to the pathogenicity of SDV in trout.

The genome of infectious hematopoietic necrosis virus (IHNV), a salmonid novirhabdovirus, has been engineered to modify the gene order and to evaluate the impact on a possible attenuation of the virus in vitro and in vivo . By reverse genetics, eight recombinant IHNVs (rIHNVs), termed NxGy according to the respective positions of the nucleoprotein (N) and glycoprotein (G) genes along the genome, have been recovered. All rIHNVs have been fully characterized in vitro for their cytopathic effects, kinetics of replication, and profiles of viral gene transcription. These rIHNVs are stable through up to 10 passages in cell culture. Following bath immersion administration of the various rIHNVs to juvenile trout, some of the rIHNVs were clearly attenuated (N2G3, N2G4, N3G4, and N4G1). The position of the N gene seems to be one of the most critical features correlated to the level of viral attenuation. The induced immune response potential in fish was evaluated by enzyme-linked immunosorbent spot assay (ELISPOT) and seroneutralization assays. The recombinant virus N2G3 induced a strong antibody response in immunized fish and conferred 86% of protection against wild-type IHNV challenge in trout, thus representing a promising starting point for the development of a live attenuated vaccine candidate. IMPORTANCE In Europe, no vaccines are available against infectious hematopoietic necrosis virus (IHNV), one of the major economic threats in fish aquaculture. Live attenuated vaccines are conditioned by a sensible balance between attenuation and pathogenicity. Moreover, nonsegmented negative-strain RNA viruses (NNSV) are subject to a transcription gradient dictated by the order of the genes in their genomes. With the perspective of developing a vaccine against IHNV, we engineered various recombinant IHNVs with reordered genomes in order to artificially attenuate the virus. Our results validate the gene rearrangement approach as a potent and stable attenuation strategy for fish novirhabdovirus and open a new perspective for design of vaccines against other NNSV.

The role of terrestrial vertebrates in the epidemiology of chikungunya disease is poorly understood. We evaluated their exposure and amplification role during the 2006 chikungunya outbreak in the Indian Ocean. Blood samples were collected from 18 mammalian and reptile species from Reunion Island, Mauritius and Mayotte. Among the 1051 samples serologically tested for chikungunya virus (CHIKV), two crab-eating macaques (Macaca fascicularis) and two ship rats (Rattus rattus) proved to be exposed to CHIKV. CHIKV RNA was not detected in 791 analyzed sera. Our results confirm the preferential infection of simian primates and suggest that other vertebrates played a poor or no role in CHIKV transmission during the 2006 outbreak.

Sleeping disease in rainbow trout is characterized by an abnormal swimming behaviour of the fish which stay on their side at the bottom of the tanks. This sign is due to extensive necrosis and atrophy of red skeletal muscle induced by the sleeping disease virus (SDV), also called salmonid alphavirus 2. Infections of humans with arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), are global causes of debilitating musculoskeletal diseases. The mechanisms by which the virus causes these pathologies are poorly understood due to the restrictive availability of animal models capable of reproducing the full spectrum of the disease. Nevertheless, it has been shown that CHIKV exhibits a particular tropism for muscle stem cells also known as satellite cells. Thus, SDV and its host constitute a relevant model to study in details the virus-induced muscle atrophy, the pathophysiological consequences of the infection of a particular cell-type in the skeletal muscle, and the regeneration of the muscle tissue in survivors together with the possible virus persistence. To study a putative SDV tropism for that particular cell type, we established an in vivo and ex vivo rainbow trout model of SDV-induced atrophy of the skeletal muscle. This experimental model allows reproducing the full panel of clinical signs observed during a natural infection since the transmission of the virus is arthropod-borne independent. The virus tropism in the muscle tissue was studied by immunohistochemistry together with the kinetics of the muscle atrophy, and the muscle regeneration post-infection was observed. In parallel, an ex vivo model of SDV infection of rainbow trout satellite cells was developed and virus replication and persistence in that particular cell type was followed up to 73 days post-infection. These results constitute the first observation of a specific SDV tropism for the muscle satellite cells.