Background

Viral haemorrhagic septicaemia virus (VHSV) is an enveloped negative single-stranded RNA virus belonging to the genus Novirhabdovirus of the Rhabdoviridae family. It is the aetiological agent of viral haemorrhagic septicaemia (VHS), one of the most important viral diseases of salmonid fish in European aquaculture with primer impact on commercial rainbow trout (Oncorhynchus mykiss) production. The virus usually causes severe hemorrhages in the skin, muscles, eyes, kidney and liver, with mortality rates up till 90%.

VHSV was first isolated and identified from farmed rainbow trout in Denmark in 1965. It was described as a freshwater salmonid disease agent only. Since then, big emphasis was put on veterinary surveillance programs to eradicate the virus with the result that Danish freshwater systems are approved to be VHS-free zones according to the European Union legislation (Council Directive 91/67/EC). However, in the last decade VHSV has been isolated from continuously increasing numbers of free-living marine fish species in waters near Europe, North America and Japan, indicating a widespread occurrence of the virus in the marine environment of the Northern hemisphere with emerging potential.

Even though, marine VHSV isolates in general are non-pathogenic for rainbow trout, they present a huge but underestimated threat to rainbow trout aquaculture. Single-stranded RNA viruses, such as VHSV, are the most rapidly evolving entities on earth, exhibiting very high mutation rates and generating new forms with almost each replication. RNA virus populations therefore often represent complex mixtures of different, but closely related variants also known as quasi-species. As a consequence, RNA viruses have a high potential for continuous adaptation to host shifts indicating, that VHSV might easily be able to evolve from a non-pathogenic to a pathogenic form when giving access to a new host. Examples of such host adaption have been observed in rainbow trout reared in brackish/marine waters in Finland, Sweden and very recently also in Norway, where newly recovered VHSV isolate closest related to existing marine VHSV isolates caused mortality in rainbow trout.

Aim

The production of rainbow trout in marine aquaculture systems is an expanding industry in Denmark and other European countries like Norway and Sweden. However, the existence of a circulating reservoir of VHSV in marine wild fish populations and the ability of RNA viruses to rapid adaptation represents an important threat towards the future success of mari-cultured rainbow trout production.

This project aims at evaluating the potential risk of marine VHSV isolates for rainbow trout in sea cage systems by:

1. Identifying further transmission routes to verify that marine VHSV isolates can achieve host entry
2. Demonstrate the process of evolution from non-pathogenic to pathogenic forms following host shift
3. Identifying viral genetic signatures in VHSV that are correlated to pathogenicity in rainbow trout

Research Outline

The existence of oral transmission routes will be analyzed to identify if natural prey fish, infected with VHSV, might pose a disease risk in mari-culture. A recombinant VHSV isolate, expressing the Renilla luciferase gene, will be used for oral infection and bioluminescence imaging techniques will be applied to visualize the entrance sites of VHSV.

Attachment and replication dynamics of marine and freshwater VHSV isolates will be compared in vivo to get an idea of the causation leading to the existing pathogenicity differences among marine and freshwater isolates. 

A serial passage experiment with a marine VHSV isolate will be performed, applying differing selection strategies, to demonstrate that increase of pathogenicity during passage through several passes of rainbow trout is possible.

Finally recovered VHSV isolates from the serial passage experiment as well as several marine and freshwater VHSV isolates will be exposed to deep sequencing analysis to identify sequence motives within the VHSV genome that are correlated to patogenicity in rainbow trout.