title: Specific nucleotides at the 3'-terminal promoter of viral hemorrhagic septicemia virus are important for virulence in vitro and in vivo.
abstract: 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.
authors.affiliation: Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Bioproduction, P.O. Box 8146 Dep, N-0033 Oslo, Norway.
authors.forename: Sung-Hyun
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Bioproduction, P.O. Box 8146 Dep, N-0033 Oslo, Norway.
authors.forename: Tz-Chun
authors.initials: TC
authors.lastname: Guo
authors.affiliation: Institute of Marine & Environmental Technology, University of Maryland Baltimore County, 701 East Pratt Street, Baltimore 21202, USA.
authors.forename: Vikram N
authors.initials: VN
authors.lastname: Vakharia
authors.affiliation: Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Bioproduction, P.O. Box 8146 Dep, N-0033 Oslo, Norway.
authors.forename: Øystein
authors.initials: Ø
authors.lastname: Evensen
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D005260:Female; D005393:Fish Diseases / Q000821:virology*; D015967:Gene Expression Regulation, Viral; D008297:Male; D008969:Molecular Sequence Data; D029141:Novirhabdovirus / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity*; D011401:Promoter Regions, Genetic; D018353:Rhabdoviridae Infections / Q000662:veterinary* / Q000821:virology; D014774:Virulence; D015027:Zebrafish
chemical_list: D020413:3' Untranslated Regions
keywords: 3′-UTR; In vitro; In vivo; Novirhabdovirus; Promoter function; VHSV; Virulence