The NV Gene of Snakehead Rhabdovirus (SHRV) Is Not Required for Pathogenesis, and a Heterologous Glycoprotein Can Be Incorporated into the SHRV Envelope

Abstract

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...

Limited Interference at the Early Stage of Infection between Two Recombinant Novirhabdoviruses: Viral Hemorrhagic Septicemia Virus and Infectious Hematopoietic Necrosis Virus

“…Thus, rVHSV-⌬NV appears to be as attenuated as previously described for rIHNV-⌬NV, confirming the essential role of the NV protein of salmonid novirhabdovirus. In contrast to our findings, Alonso et al, using a reverse-genetic system established with snakehead rhabdovirus (SHRV), a warm-water fish novirhabdovirus, have observed no apparent role of the NV protein in replication in vitro as well as in fish pathogenesis (1). It could be of interest to replace the VHSV and IHNV NV genes with that of SHRV to evaluate the potential of the complementation of this NV protein on both virus phenotypes and thus evaluate whether these three NV proteins that share less than 30% of homology have a common function.…”

Identifying potential virulence determinants in viral haemorrhagic septicaemia virus (VHSV) for rainbow trout

“…In addition to SE-SVA-1033, isolate DK-5e59 was the only remaining isolate to encode a Phe at this point mutation. Due to the relatively small size of the viral genome (approximately 11 000 nt), Alonso et al (2004) postulated the NV gene (369 nt) must have an important role in viral replication, although it was not essential for pathogenesis in snakehead rhabdovirus (SHRV). However, Thoulouze et al (2004) showed the gene to be essential for pathogenicity of another salmonid rhabdovirus, infectious haematopoietic necrosis virus (IHNV), whereby removal of the NV gene significantly reduced the virulence of the virus for rainbow trout, as also demonstrated by Brémont (2005).…”

“…VHSV is a rhabdovirus comprised of a single-stranded RNA genome encoding nucleo-(N), phospho-(P), matrix-(M), glyco-(G) and RNA-dependent (L) polymerase structural proteins. Classified within the genus Novirhabdoviridae, VHSV also encodes a characteristic non-structural (non-virion, NV) protein, situated between the G and L genes, the functional role of which remains uncertain (Alonso et al 2004. Four main genotypes of the virus have been identified which are independent of the genomic region used for analysis (EinerJensen et al 2004) and are restricted to 4 broadly distinct geographical locations (Snow et al 2004).…”

Characterization of Snakehead Rhabdovirus Infection in Zebrafish ( Danio rerio )

“…Natural SHRV infection has only been associated with the complex disease EUS; however, experimental infection with SHRV has been successful in snakehead fry and juveniles and in zebrafish (2,12,21,29). This is the first study to examine, in detail, the pathogenesis and antiviral response in zebrafish associated with SHRV infection.…”

“…Zebrafish infected with SVCV exhibited significant mortalities and histopathology, marked by necrosis of the gills, liver, and spleen. In a recent collaboration between our lab and the Leong lab (2), recombinant SHRV mutants were used to demonstrate the role of the SHRV nonvirion gene during infection in adult zebrafish; however, detailed pathogenesis and antiviral response were not reported.…”