Production of biologically active Atlantic salmon interferon in transgenic potato and rice plants.

Interferons (IFNs) are cytokines that induce an antiviral state in vertebrate cells. The Atlantic salmon (Salmo salar) IFN gene (SasaIFN-alpha1) was introduced in potato and rice plants by Agrobacterium-mediated transformation to produce a biologically active fish IFN in these plants. The transgenes and their transcripts were detected by PCR and Northern blot analysis. Western blot analysis showed the existence of SasaIFN-alpha1in transgenic plants. The antiviral activity of the SasaIFN-alpha1 protein expressed in these plants was determined by the survival rates of pre-treated cultured fish cells against pancreatic necrosis virus infection. The survival rate of cells pre-treated with transgenic samples was up to 95% but was reduced to 30-47% when cells were pre-treated with non-transgenic samples. These results demonstrated an antiviral effect of the SasaIFN-alpha1 protein derived from transgenic plants. Plant-derived IFNs may be suitable as components of functional feeds because such IFNs are free of animal pathogens and can be produced at a lower cost compared with those from transgenic mammalian and bacterial cells. This is the first study describing the production of a biologically active fish IFN using transgenic plants.

Screening of freshwater fish species for their susceptibility to a betanodavirus.

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. Because the genomes are the smallest and simplest among viruses, betanodaviruses have been well studied using a reversed genetics system as model viruses. However, studies of virus-host interactions have progressed slowly because permissive hosts for betanodaviruses (basically larvae and juveniles of marine fish) are only available for limited periods of the year and are not suitable for the construction of a genetic engineering system. To obtain a model fish species that are not subject to these problems, 21 freshwater fish species were injected intramuscularly with a betanodavirus (redspotted grouper nervous necrosis virus) and tested for their susceptibility to the virus. Based on their responses, the tested fish were classified into 3 groups: 4 susceptible fish, 10 less susceptible fish, and 7 resistant fish. The susceptible fish, celebes rainbowfish Telmatherina ladigesi, threadfin rainbowfish Iriatherina werneri, dwarf rainbowfish Melanotaenia praecox, and medaka Oryzias latipes, exhibited erratic swimming and eventually died within 10 d post-inoculation. The virus was specifically localized in the brains, spinal cords, and retinas of the infected fish, similar to the pattern of infection in naturally infected marine fish. We believe that these susceptible freshwater fish species could act as good host models for betanodavirus-fish interaction studies.

Betanodavirus infection in the freshwater model fish medaka (Oryzias latipes).

Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite, positive-sense RNA genomes. As their genomes are the smallest and simplest among viruses, betanodaviruses have been studied in detail as model viruses by using a genetic-engineering system, as has occurred with the insect alphanodaviruses, the other members of the family Nodaviridae. However, studies of virus-host interactions have been limited, as betanodaviruses basically infect marine fish at early developmental stages (larval and juvenile). These fish are only available for a few months of the year and are not suitable for the construction of a reverse-genetics system. To overcome these problems, several freshwater fish species were tested for their susceptibility to betanodaviruses. It was found that adult medaka (Oryzias latipes), a well-known model fish, was susceptible to both Striped jack nervous necrosis virus (the type species of the genus Betanodavirus) and Redspotted grouper nervous necrosis virus (RGNNV), which have different host specificities in marine fish species. Infected medaka exhibited erratic swimming and the viruses were localized specifically in the brain, spinal cord and retina of the infected fish, similar to the pattern of infection in naturally infected marine fish. Moreover, medaka were susceptible to RGNNV at the larval stage. This is the first report of a model virus-model host infection system in fish. This system should facilitate elucidation of the mechanisms underlying RNA virus infections in fish.