Salmonid alphavirus (SAV) is an enveloped, single-stranded, positive-sense RNA virus with a ~12 kb genome, belonging to the family
The SAV genome is a single-stranded, positive-sense RNA with two open reading frames (ORFs). It encodes eight proteins: four structural capsid glycoproteins (E1, E2, E3, and 6K) by the 3′ end and four nonstructural proteins (nsP1-4) by the 5′ end (38). Protein E2 has an important function in receptor binding to the host cell, mediating the internalisation of the virus and playing a major role in the final envelope formation and in the budding of new viruses (10). Moreover, most neutralising epitopes are present on the E2 glycoprotein, while E1 protein contains more conserved, cross-reactive epitopes (26). Six subtypes of salmonid alphavirus (SAV1–6) have been created (Table 1) based on the nucleic acid sequences of genes encoding E2 and nsP3. A study on the serological cross-reactivity between the different subtypes of SAV was conducted by Graham
Summary of salmon alphavirus (SAV) subtypes PD – pancreas disease SD – sleeping disease
Virus subtype
Optional virus name
Disease
Species
Location
SAV1
Salmon pancreas disease virus
PD
Atlantic salmon (sea water) Rainbow trout (fresh water
Ireland, Scotland
SAV2 FW
Sleeping disease virus
SD
Rainbow trout (fresh water) Atlantic salmon (sea water)
France, England, Scotland, Germany, Italy, Spain, Poland, Switzerland
SAV2 MW
Salmon pancreas disease virus
PD
Atlantic salmon (sea water)
Norway, Scotland
SAV3
Norwegian salmon alphavirus
PD
Atlantic salmon (sea water) Rainbow trout (fresh water)
Norway
SAV4
Salmon pancreas disease virus
PD
Atlantic salmon (sea water)
Ireland, Scotland
SAV5
Salmon pancreas disease virus
PD
Atlantic salmon (sea water)
Scotland
SAV6
Salmon pancreas disease virus
PD
Atlantic salmon (sea water)
Ireland
Salmonid alphavirus subtype 1 (SAV1) has been described as the causative agent of pancreas disease in Atlantic salmonids in Ireland and Scotland (14, 27). Sleeping disease in rainbow trout is known to be caused by the second subtype of SAV (SAV2). SD has been detected in France (6, 8), England and Scotland (11), and Germany (4). It has been also reported in Italy and Spain (13) and more recently in Poland (5) and Switzerland (30). SAV2 has been divided into two subgroups (Table 1) named freshwater variant (SAV2 FW) and marine variant (SAV2 MW). Infections with SAV2 FW cause sleeping disease in freshwater-reared rainbow trout in England, Scotland, and mainland European countries (21). The marine variant of SAV2 (SAV2 MW) is responsible for pancreas disease in seawater-reared Atlantic salmon. The third subtype of salmonid alphavirus (SAV3) has been isolated from farmed Atlantic salmon in Norway (17), hence it is also named Norwegian salmonid alphavirus (NSAV). SAV3 causes pancreas disease in Atlantic salmon and sea-reared rainbow trout. It has been shown that SAV3 has genomic organisation identical to that of SAV1 and SAV2 (26). To date, SAV3 has only been detected in Norway, where until 2010 no other subtypes were detected (17, 20). Subtypes 4–6 of SAV (SAV4–6) have been detected in Scotland and Ireland in connection with PD outbreaks (10) and detected there along with SAV1 (14).
The main transmission way of salmonid alphavirus appears to be horizontal and
Pancreas disease is an infectious disease affecting specifically Atlantic salmon and it was first detected in Scotland in the 1970s. Later, there were reports of the disease in farmed Atlantic salmon from Ireland and Norway. There were no reports of PD outbreaks outside Europe but for one exception when the disease was described in North America (without detection a virus) (21).
SAV1 can be isolated in all regions of Ireland, which makes it the dominant SAV subtype in this country. However, SAV4 can also be found in northwest Ireland. In Norway, more subclinical cases of PD are caused by marine SAV2 infections than by SAV3 (21). There is also a study suggesting that about a third of populations infected by SAV may not develop clinical PD (1).
PD usually occurs in salmon smolts during the first year at sea but recently there have been more reports showing that other year classes of Atlantic salmon are also susceptible. Outbreaks are common between late July and early September (26). Clinical signs associated with PD include sudden inappetence, lethargy and an increased number of faecal casts in the cages, increased mortality and ill-thrift (25, 26). The histopathological changes in fish affected by PD and SD primarily occur in the pancreas, heart, and skeletal muscles. For many years, the descriptions of PD from Scotland and Ireland (reviewed by McLoughlin
Sleeping disease (SD) is an infectious disease similar to pancreas disease; however, it affects rainbow trout reared in fresh water. It was first observed in France (6) and the virus (SDV) was isolated a few years later (8).
The causative agents of SD and PD are closely related members of the same species, generally known as salmonid alphavirus, which has been proven by complete genome sequencing (38). The causative agent of SD is the subtype 2 (SAV2) (37).
SD is an increasing problem not only in France but throughout Europe, causing higher mortality and growth retardation of fish. It is known to affect rainbow trout at all stages of production (26). SAV2 has been isolated in England and Scotland (11), in Italy and Spain (13), and in Germany (4). More recently, the first laboratory confirmation of SAV2 infection in Poland has been described (5), and the first outbreak of sleeping disease in Switzerland has been reported (30).
The characteristic sign of sleeping disease is the unusual behaviour of affected fish, manifested in their laying on their side on the bottom of the tank, hence the name “sleeping” disease. Extensive necrosis of skeletal red muscles is considered to be the cause of this behaviour. This chronic stage follows the development of characteristic sequential histological lesions of the exocrine pancreas and heart (6).
Initially, the diagnosis of PD and SD was based on clinical signs combined with typical histopathological findings. Currently, a combination of histopathological examination, antibody detection, virus culture, and PCR technique is used. Detailed descriptions of SAV diagnostic methods are contained in the Manual of Diagnostic Tests for Aquatic Animals published by the OIE (19). The first successful isolation of SAV1 was described by Nelson
However, since methods based on antibody detection and virus isolation are time-consuming and have some limitations, one such being that not all SAV isolates from clinical samples can be cultured in fish cells
Braceland
Based on the field observations showing that fish surviving SAV infections were resistant to reinfection, it has been suggested that vaccination may be applied for control of PD and SD. The results of the study by Graham
There are a few commercial vaccines against PD available. The PD vaccine produced by Pharmaq (Norway) and the monovalent Norvax Compact PD (NCPD) vaccine (MSD Animal Health, Norway) are based on an inactivated PD virus. Multivalent vaccines (the three-component AquaVac PD3 in Ireland and Scotland and the seven-component AquaVac PD7 in Norway) have been commercially available since 2015 and are also based on inactivated viruses (21) (Table 2).
Commercial vaccines against salmonid alphavirus
Vaccine name
Company
Type
Delivery method
Target species
PD
Pharmaq AS Norway
inactivated
injection
Atlantic salmon
Norvax Compact PD
MSD Animal Health
inactivated
injection
Atlantic salmon
AquaVac PD3
MSD Animal Health
inactivated
injection
Atlantic salmon
AquaVac PD7
MSD Animal Health
inactivated
injection
Atlantic salmon
In the case of the Norvax Compact PD vaccine a reduction in mortality of at least 50% is claimed in comparison of vaccinated fish against unvaccinated fish at the same farm (3). The Intervet Norbio vaccine showed reduction in histopathological damages (3, 21). It has been used in Norwegian aquaculture of salmon since 2007. A cohort study of the effect of vaccination on pancreas disease (3) showed the positive effects of the vaccination protocol against PD in Norwegian salmon farming. Vaccination has reduced the number of outbreaks and has decreased cumulative mortality and the number of discarded fish at slaughter as well as increased the growth rate compared to non-vaccinated fish.
On the other hand, the annual number of SAV3 outbreaks in Norway is still high, thus the prevention from PD provided by the Norvax Compact PD vaccine is questionable (9). Therefore, it was necessary to develop more effective alternatives, for example DNA vaccines that showed a high level of protection against rhabdoviruses in salmonids. In DNA vaccination it is not the antigen itself being administrated but the gene encoding the vaccine agent. Further antigen expression by cells in the vaccinated hosts triggers the host immune system. In a recent study (9) researchers compared the efficacy of two DNA vaccine constructs against SAV3 (pCSP and pE2) and a commercial vaccine based on inactivated SAV1 (Norvax Compact PD (NCPD)). The pCSP construct was a plasmid expressing the whole SAV3 structural polyprotein C-E3-E2-6K-E1, while plasmid pE2 encoded the SAV3 surface protein E2 alone. The study showed that vaccination with both pCSP and NCPD reduced the virus numbers in serum. The histopathological findings demonstrated that pCSP provided superior protection against SAV3 infections compared to NCPD and pE2. It was confirmed that the commercial NCPD vaccine increased protection against SAV3, but the results also revealed that it failed to protect fish against development of pathological lesions in the pancreas and heart. The authors claimed that it might partly explain the inability of NCPD to diminish outbreaks of SAV in Norwegian salmon farming. However, it was not clarified whether the inferior effect of NCPD on antibody production and protection compared to pCSP was due to its SAV1 derivation being second-best to pCSP’s SAV3. However, E2 from SAV1 and this protein from SAV3 shows very high amino acid sequence identity (95%).
It has also been demonstrated in experimental trials that both faeces and mucus are involved in the shedding of SAV from infected salmon (15). Andersen
Salmonid alphavirus (SAV) infections have been known since the 1980s and have become economically significant salmonid diseases. Many studies have been performed to advance the knowledge of SAV and the disease itself, yet there are still a number of areas that require further research, such as transmission of the virus and possible risk related to the export of SAV-infected fish. Currently, fast and sensitive PCR-based techniques are in place for virus detection; however, they should not be used as the sole diagnostic indicator. A combination of histopathological examination, antibody detection, virus culture, and PCR technique should be used for diagnosis of SAV infections. As SAV infections are a large problem with economic importance, it is clear that aside from fast virus detection, prophylaxis against SAV is needed. Field observations showed that fish surviving SAV infections were resistant to reinfection, which made the case for development of vaccines with positive effects in controlling SAV diseases.