Studies of mechanisms of virulence of viral hemorrhagic septicemia (VHS) : comparison between different genotypes of the virus

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 highand 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. IMPORTANCE This is the first study to clearly link in vitro virulence of viral hemorrhagic septicemia virus (VHSV) with an amino acid residue in the L protein, a site located in conserved region IV of the L protein. In vitro virulence is documented by induction of cytopathic effects and viability studies of gill epithelial cells, and the observed cellular responses to infection are associated with increased viral replication levels. There are no previous studies addressing the importance of the L protein or the RNA-dependent RNA polymerase for virus virulence in vitro or in vivo. Therefore, the findings reported here should broaden the search for pathogenicity traits in novirhabdoviruses, and there is a possibility that the polymerase participates in defining the host species virulence of various VHSV strains. Viral hemorrhagic septicemia virus (VHSV) is an enveloped, nonsegmented negative-sense (NNS), single-stranded RNA virus that belongs to the Novirhabdovirus genus of the family Rhabdoviridae (1). The virus genome contains the nucleocapsid (N) protein, polymerase-associated phosphoprotein (P), matrix (M) protein, surface glycoprotein (G), and viral polymerase (L) genes and a nonstructural (NV) protein gene (1). Until the late 1980s, VHSV was thought to cause only a disease of farmed rainbow trout (Oncorhynchus mykiss) in Europe, but with the detection of VHSV in returning Chinook (2) and Coho (3) salmon in the United States, later defined as genotype IVa, its detection in Atlantic cod (Gadus morhua) (4), and later the identification of a VHSV (genotypes I to III) in a vast number of marine fish species in Europe (5), the understanding of the virus’s origin, its species range, and the dynamics of the interchange between wild and farmed populations changed. Investigations into the source of the trout-pathogenic European genotype Ia isolate showed that it likely emerged from a marine source some 60 years earlier (6). Today, VHSV is separated into four different genotypes (I to IV) with different sublineages (6, 7), and the different genotypes are separated geographically. All European marine VHSV strains have been shown to be nonpathogenic or have very low pathogenicity to rainbow trout after a waterborne challenge (5, 6, 8). The marine source of genotype I (6–11) and the later emergence of a troutvirulent genotype III isolate of marine origin in Norway (12) show that VHSV is capable of crossing host species barriers. Obviously, this creates a continuous concern to aquaculture operations, but it has not been possible to link virulence traits to specific sequence motifs of any of the expressed virus proteins. One approach has Received 16 February 2014 Accepted 6 April 2014 Published ahead of print 9 April 2014 Editor: D. S. Lyles Address correspondence to Øystein Evensen, oystein.evensen@nmbu.no. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00423-14 July 2014 Volume 88 Number 13 Journal of Virology p. 7189 –7198 jvi.asm. org 7189 on Jne 5, 2014 by N O R G E S V E T E R IN A E R H O G S K O LE http/jvi.asm . rg/ D ow nladed fom