Keynote-3
APPLICATION OF A GENETIC-ENGINEERING SYSTEM FOR THE STUDY OF BETANODAVIRUS
(“tricks-of-the-trade” the application of reverse genetics)
Tokinori Iwamoto*
Department of Pathology & Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Canada
*
The lack of basic RNA processing technology has long hampered direct gene manipulation of the RNA viral genome. For example, the use of reliable techniques such as site directed-mutagenesis to produce precisely defined nucleotide substitution in a RNA molecule has not been possible. Subsequent to the discovery of the positive-sense RNA virus infectious cDNA clone system, however, the DNA molecule has become a vehicle for manipulation of the genome of RNA viruses. A direct consequence of these advances in recombinant DNA technology has been the establishment of “reverse genetics systems”, leading to accelerated study of RNA viruses and their interaction with host.
Ten years after the first published report of striped jack nervous necrosis virus (SJNNV), the type species of the genus Betanodavirus, a reverse genetic system for betanodaviruses was initially established in SJNNV. Subsequently, these methodologies have been applied to the other betanodavirus clade for the investigation of the role of the virus RNA molecules and proteins in replication, pathogenesis and host-specificity. In this presentation, an overview of reverse genetics system for SJNNV and examples of the application of these techniques in the study of RNA replication and host-specificity determinants of betanodaviruses will be discussed.
Establishment of a reverse genetics systems for SJNNV (PMID: 11602777)
The complete 5’- and 3’-terminal sequences of SJNNV genomic RNA1 and RNA2 were determined, and SJNNV full-length cDNA clones for infectious RNA transcription were then constructed. Although the infectivity of the in vitro transcripts mixture of RNA1 and RNA2 in E-11 cells was considerably low when compared to the authentic SJNNV virion RNAs, the progeny viruses that were generated caused mortality of striped jack larvae. This finding demonstrated the application of recombinant DNA technology to manipulate SJNNV genome.
A study of virus-host interaction by using reassortant viruses between SJNNV & SGNNV (PMID: 14722280)
To identify host-specificity determinants in betanodaviruses, construct viruses that contained reassorted genomes between SJNNV and Sevenband grouper nervous necrosis virus (SGNNV) were recovered from in vitro transcript RNA-transfected E-11 cells. Subsequent experimental infection of the reassorted viruses against the original host species resulted in the determination that viral RNA2 and/or the encoded coat protein controls host-specificity in these viruses.
A site-directed mutagenesis study of SJNNV RNA3 (PMID: 16186236)
A small subgenomic RNA3, which corresponds to the 3'-terminal region of SJNNV RNA1, and RNA3-encoded protein (B2), can be detected in virus-infected cells. Single-nucleotide substitutions (T to G, T to C, and T to A) at the position of 5’ end of RNA3 in a RNA1 cDNA clone resulted in diminishing of positive-sense RNA3 accumulation in transfected E-11 cells. Similarly, production of the B2 was strongly inhibited by an amino acid substitution introduced into the initiation and the stop codons. Evaluations of SJNNV B2 protein using an Agrobacterium co-infiltration assay in plants suggest that this protein has a potent RNA silencing-suppression activity.