Characterisation of ISA HPR0 virus- and infection in Atlantic salmon

At start-up of this project, it was a commonly accepted hypothesis that disease-causing (virulent) ISA viruses are evolving from non-virulent ISA HPR0 virus. In the current project, we have combined pathology, virology, and molecular biology to describe, and characterize ISA virus infection in Atlantic salmon with the non-virulent HPR0 type, in particular comparing the infection with classical ISA. A major goal was to identify functions that were different and thus could help identifying virulence factors for ISA virus in general. To increase the knowledge regarding these virus types would also enable us to evaluate the risk of ISA outbreaks when the HPR0 type is detected, and to identify drivers for this evolution. Early in the project, we presented results from an extensive PCR screening of Atlantic salmon gills on the Faroe Islands. Here we found that +/- 10% of all samples were HPR0 positive, and that all sites experienced a transient ISA HPR0 infection in the seawater production phase. Later we have demonstrated that some freshwater smolt farms had a nearly similar ISA HPR0 prevalence. In one year, close to all brood fish gills were HPR0 positive at stripping, however, we found no association between these findings and HPR0 findings in smolts. Furthermore, we also demonstrated through immunohistochemical, immunofluorescence, and PCR analyses of tissue from ISA HPR0 virus infected fish, that ISA HPR0 virus cause a superficial and localized infection of mucosal surfaces. We also showed that HPR-del virus had a similar tissue distribution early in the infection phase before the infection become a generalized infection of endothelial cells of the blood circulatory system. A new method for detecting the ISA virus cellular receptor in tissue sections was also developed, and it was shown during our study that distribution of the receptor, and modulation of the viral fusion activity (see below) determined cell, and tissue tropism. The receptor detection method was also used to screen more than 50 different fish species of which 33 expressed the receptor, which is a necessary requirement for the ISA virus infection to establish. Thus these fish may potentially be infected by ISA virus. In the winter of 2014, HPR-del virus was detected at the Faroe Islands for the first time since the devastating ISA epidemic ending in 2005. The new virus was detected during routine PCR screening at slaughter, and no typical clinical and pathological signs suggesting ISA were observed. We were, however, able to identify the HPR0 source of the new HPR-del virus in the smolt farm where the affected fish came from. Further investigation including full genome sequencing of the two viral genomes, and infection experiments, revealed the only differences were the HPR deletions and one mutation in the F-protein. Experimental infections revealed a low virulent virus, but with a similar tissue distribution as found with highly virulent ISA virus. Thus, these changes were found to be a minimum requirement for shift from localized HPR0 infection, to a generalized HPR-del infection. In this study we also suggest that the evolution from non-virulent ISA HPR0 virus to highly virulent HPR-del virus, is a stepwise process involving low virulent intermediates that may be difficult to detect by currently used surveillance procedures. Laboratory experiments where ISA virus HE-, and F-proteins were expressed in cell cultures through a method called transfection, revealed that these two protein collaborated in providing the viral fusion activity. Fusion activity is essential for virus uptake when cells become infected, and is a key factor for viral cell tropism. Our studies showed that deletions of the HPR in combination with mutations in the F-proteins, both affected fusion activation, and activity. The virus quasispecies hypothesis says that virus appears in swarms, usually dominated by one virus type, but with numerous genetic variants that ensures virus to rapidly respond to changes in the environment. Quasispecies spectra were investigated in both ISA HPR0 and HPR-del samples using Next generation sequencing. We did not find neither related HPR-del virus in samples dominated by ISA HPR0 virus, nor related HPR0 virus in samples dominated by HPR-del virus. Cultivation of HPR0 virus in cell cultures has so far been unsuccessful. To be able to do so would have been very helpful both to examine and characterize factors associated with the evolution from HPR0 to HPR-del virus. Several unsuccessful attempts were done to replicate ISAV HPR0 virus in cell cultures. This included the establishment of both primary cell cultures, and new continuous cell cultures from Atlantic salmon gill and skin. However, at the end of this project new knowledge had been established pointing out the direction to go to be able culture this virus in the future, though some basic work needs to be done first.

Infectious salmon anaemia (ISA) is still a significant problem for the aquaculture industry in Norway due to direct losses of Atlantic salmon caused by disease outbreaks and the measures taken to control the infection. The severity of the disease is empha sized by the large ISA epidemics unfolding over the past two years within a small geographical region in the county of Troms, having great impact on local economy. In this project we will be investigating molecular-, functional-, pathological- and evoluti onary properties associated with low- or avirulent ISA virus (ISAV) HPR0 genotypes, assumed to be precursors of all virulent ISAV strains that cause classical ISA outbreaks. The main objective through this multidisciplinary approach will be to obtain new information that can aid in forming the necessary basis for evaluating important issues related to HPR0, like the risk of infection and its frequency of transition to virulence. Both the authorities and industry request more information concerning this is sue in order to be able to take appropriate protective measures, especially as recent findings have shown there is a higher HPR0 prevalence in the marine environment than previously assumed, thus causing interpretation problems related to ISAV screening. The investigations will span both genetic and functional aspects related to HPR0, focusing on determining factors that are required for virulence acquisition. Modified existing methodology, but also powerful new technological approaches, will be used to r each our objective. The project group has a large and increasing number of HPR0 positive samples at their disposal, both from Norway and the Faeroe Islands, providing a solid foundation for these studies. The results from the project will increase our kno wledge on ISAV genetic markers involved in virulence, and may provide a new basis for the development of genetically engineered vaccines against ISA.