1 National Veterinary Institute, Technical University of Denmark2 Section for Virology, National Veterinary Institute, Technical University of Denmark3 Norwegian University of Life Sciences4 Aarhus University
Viral hemorrhagic septicemia virus (VHSV) is separated into four different genotypes (I to IV) with different sublineages (K. Einer-Jensen, P. Ahrens, R. Forsberg, and N. Lorenzen, J. Gen. Virol. 85: 1167-1179, 2004; K. Einer-Jensen, J. Winton, and N. Lorenzen, Vet. Microbiol. 106: 167-178, 2005). European marine VHSV strains (of genotypes I to III) are, in general, nonpathogenic or have very low pathogenicity to rainbow trout after a waterborne challenge, and here we also show that genotype IVa is nonpathogenic to trout. Despite several attempts, it has not been possible to link genomic variation to in vivo virulence. In vitro virulence to gill epithelial cells (GECs) has been used as a proxy for in vivo virulence, and here we extend these studies further with the purpose of identifying residues associated with in vitro virulence. Genotype Ia (DK-3592B) and III (NO/650/07) isolates, which are pathogenic to rainbow trout (O. B. Dale, I. Orpetveit, T. M. Lyngstad, S. Kahns, H. F. Skall, N. J. Olesen, and B. H. Dannevig, Dis. Aquat. Organ. 85: 93-103, 2009), were compared to two marine strains that are nonpathogenic to trout, genotypes Ib (strain 1p8 [H. F. Mortensen, O. E. Heuer, N. Lorenzen, L. Otte, and N. J. Olesen, Virus Res. 63: 95-106, 1999]) and IVa (JF-09). DK-3592 and NO/650/07 were pathogenic to GECs, while marine strains 1p8 and JF-09 were nonpathogenic to GECs. Eight conserved amino acid substitutions contrasting high-and low-virulence strains were identified, and reverse genetics was used in a gain-of-virulence approach based on the JF-09 backbone. Mutations were introduced into the G, NV, and L genes, and seven different virus clones were obtained. For the first time, we show that a single amino acid mutation in conserved region IV of the L protein, I1012F, rendered the virus able to replicate and induce a cytopathic effect in trout GECs. The other six mutated variants remained nonpathogenic.
Journal of Virology, 2014, Vol 88, Issue 13, p. 7189-7198
VIROLOGY; ONCORHYNCHUS-MYKISS; FISH RHABDOVIRUS; VACCINE CANDIDATE; PRIMARY CULTURES; NORTH-SEA; VHSV; TEMPERATURE; INFECTION; PROTEINS; DOMAINS; Pathogenesis and Immunity