Viral hemorrhagic septicemia virus (VHSV), a member of the Novirhabdovirus genus, contains an 11-nucleotide conserved sequence at the terminal 3'- and 5'-untranslated regions (UTRs) that are complementary. To study the importance of nucleotides in the 3'-UTR of VHSV for replication of novirhabdoviruses, we performed site-directed mutagenesis of selected residues at the 3'-terminus and generated mutant viruses using a reverse genetics approach. Assessment of growth kinetics and in vitro real-time cytopathogenicity studies showed that the order of two nucleotides (A4G5) of the 3'-terminus of VHSV directly affects growth kinetics in vitro. The mutant A4G-G5A virus has reduced total positive-strand RNA synthesis efficiency (51% of wild-type) at 48h post-transfection and 70h delay in causing complete cytopathic effect in susceptible fish cells, as compared to the WT-VHSV. Furthermore, when the A4G-G5A virus was used to challenge zebrafish, it exhibited reduced pathogenicity (54% lower end-point mortality) compared to the WT-VHSV. From these studies, we infer that specific residues in the 3'-UTR of VHSV have a promoter function and are essential to modulate the virulence in cells and pathogenicity in fish.
Keywords: 3′-UTR; In vitro; In vivo; Novirhabdovirus; Promoter function; VHSV; Virulence.
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