No abstract

The zebrafish, Danio rerio, has become recognized as a valuable model for the study of development, genetics, and toxicology. Recently, the zebrafish has been recognized as a useful model for infectious disease and immunity. In this study, the pathogenesis and antiviral immune response of zebrafish to experimental snakehead rhabdovirus (SHRV) infection was characterized. Zebrafish 24 h postfertilization to 30 days postfertilization were susceptible to infection by immersion in 10 6 50% tissue culture infective doses (TCID 50 ) of SHRV/ ml, and adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection of 10 5 TCID 50 of SHRV/ ml. Mortalities exceeded 40% in infected fish, and clinical presentation of infection included petechial hemorrhaging, redness of the abdomen, and erratic swim behavior. Virus reisolation and reverse transcription-PCR analysis of the viral nucleocapsid gene confirmed the presence of SHRV. Histological sections of moribund embryonic and juvenile fish revealed necrosis of the pharyngeal epithelium and liver, in addition to congestion of the swim bladder by cell debris. Histopathology in adult fish injected i.p. was confined to the site of injection. The antiviral response in zebrafish was monitored by quantitative real-time PCR analysis of zebrafish interferon (IFN) and Mx expression. IFN and Mx levels were elevated in zebrafish exposed to SHRV, although expression and intensity differed with age and route of infection. This study is the first to examine the pathogenesis of SHRV infection in zebrafish. Furthermore, this study is the first to describe experimental infection of zebrafish embryos with a viral pathogen, which will be important for future experiments involving targeted gene disruption and forward genetic screens.

Snakehead rhabdovirus (SHRV) affects warm-water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its "nonvirion" (NV) gene. To examine the function of the NV gene, we used a recently developed reverse genetic system to produce a viable recombinant SHRV carrying an NV gene deletion. The recombinant virus was produced at the same rate and same final concentrations as the wild-type virus in cultured fish cells in spite of the NV gene deletion. The role of the NV protein in fish pathogenesis was also investigated. Zebra fish (Danio rerio) were infected with the NV deletion mutant or with a recombinant virus containing a copy of the SHRV genome, and similar mortality rates as well as final mortalities were recorded, suggesting no apparent role for the NV protein in fish pathogenesis. Interestingly, the unsuccessful rescue of fully viable recombinants with genomes containing deletions in the G/NV gene junction suggested a role for the gene junction in virus transcription and replication. Finally, we demonstrated that the SHRV glycoprotein can be replaced by the glycoprotein of infectious hematopoietic necrosis virus (IHNV) or by a hybrid protein composed of SHRV and IHNV sequences.Snakehead rhabdovirus (SHRV), a rhabdovirus of warmwater fish, was isolated from a diseased snakehead fish (Ophicephalus striatus) during an epizootic outbreak in Thailand (12). The disease was characterized by a severe ulcerative dermal necrosis and was seen in a wide variety of freshwater and estuarine fishes, in the wild and in pond culture, throughout Southeast Asia. Although the disease affected several species, the cultured snakehead fish, the walking catfish (Clarius batrachus), and the sand goby (Oxyeleotris marmoratus) were the most noticeably affected (10). SHRV is a nonsegmented negative-strand RNA virus from the Rhabdoviridae family, genus Novirhabdovirus. The genome of SHRV contains six open reading frames (ORF) in the order 3Ј-N-P-M-G-NV-L-5Ј (13). At the junction between two genes, the stop/polyadenylation signal and the following start signal are separated by an intergenic region composed of several nucleotides that are not present in the mRNAs. The major distinguishing feature of the Novirhabdoviruses is the presence of a nonvirion (NV) gene between the viral glycoprotein (G) and the polymerase (L) genes (13). The NV gene contains a single ORF, which is 335 nucleotides long. The NV protein has been detected in infectious hematopoietic necrosis virus (IHNV)-infected tissue culture cells by autoradiography (14) and immunofluorescence techniques (21) and in tissues of IHNV-infected fish by confocal microscopy (6). While the functions of most IHNV proteins have been determined, the function of the NV protein remains unknown.In 2000, we reported the recovery of recombinant snakehead rhabdovirus (SHRV) from a full-length cDNA clone of the viral genome (11). The full-length cDNA was assembled from clones of each of the five SHRV genes and their intergenic regions and inserted into a transcription plasmid...