Abstract
Viral hemorrhagic septicemia virus (VHSV) is a highly contagious virus leading to high mortality in a large panel of freshwater and marine fish species. VHSV isolates originating from marine fish show low pathogenicity in rainbow trout. The analysis of several nearly complete genome sequences from marine and freshwater isolates displaying varying levels of virulence in rainbow trout suggested that only a limited number of amino acid residues might be involved in regulating the level of virulence. Based on a recent analysis of 55 VHSV strains, which were entirely sequenced and phenotyped in vivo in rainbow trout, several amino acid changes putatively involved in virulence were identified. In the present study, these amino acid changes were introduced, alone or in combination, in a highly-virulent VHSV 23–75 genome backbone by reverse genetics. A total of 35 recombinant VHSV variants were recovered and characterized for virulence in trout by bath immersion. Results confirmed the important role of the NV protein (R116S) and highlighted a major contribution of the nucleoprotein N (K46G and A241E) in regulating virulence. Single amino acid changes in these two proteins drastically affect virus pathogenicity in rainbow trout. This is particularly intriguing for the N variant (K46G) which is unable to establish an active infection in the fins of infected trout, the main portal of entry of VHSV in this species, allowing further spread in its host. In addition, salmonid cell lines were selected to assess the kinetics of replication and cytopathic effect of recombinant VHSV and discriminate virulent and avirulent variants. In conclusion, three major virulence markers were identified in the NV and N proteins. These markers explain almost all phenotypes (92.7%) observed in trout for the 55 VHSV strains analyzed in the present study and herein used for the backward validation of virulence markers. The identification of VHSV specific virulence markers in this species is of importance both to predict the in vivo phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.
Highlights
Viral hemorrhagic septicemia (VHS) is a very contagious viral disease leading to high mortality in young cultured and wild fish worldwide, and is listed as notifiable by the World Organization for Animal Health (OIE) (Skall et al, 2005; Walker and Winton, 2010)
Unique restriction enzyme sites found in Viral hemorrhagic septicemia virus (VHSV) 23–75 infectious cDNA (Biacchesi et al, 2010) were used to subclone VHSV genomic fragments in which nucleotide substitutions were inserted by site-directed mutagenesis
Almost all variants reached high titers between 1 × 108 to 1 × 109 PFU/mL similar to that obtained by recombinant VHSV (rVHSV) (1 × 109 PFU/mL)
Summary
Viral hemorrhagic septicemia (VHS) is a very contagious viral disease leading to high mortality in young cultured and wild fish worldwide, and is listed as notifiable by the World Organization for Animal Health (OIE) (Skall et al, 2005; Walker and Winton, 2010). VHS is one of the fish viral diseases considered by the OIE as a serious economic and social threat for fish farms with significant environmental impact on natural resources and endangered fish species. Mortality varies depending on several environmental and physiological factors such as fish age, rearing condition, stress, fish species, virus strain, water temperature. The virus reservoirs are infected domestic or wild fish and survivor fish of epizootic outbreaks that can become long-term carriers (Wolf, 1988; Anonymous, 2019). Fins were shown to be the main portal of entry for a closely-related virus, the Infectious Hematopoietic Necrosis Virus (IHNV) (Harmache et al, 2006)
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