# Recent findings in the molecular biology of Salmonid
Alphavirus

Michel Brémont

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## To cite this version:

Michel Brémont. Recent findings in the molecular biology of Salmonid Alphavirus. Bulletin of the European Association of Fish Pathologists, 2016, 36 (4), pp.145-150. hal-02630429

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SHORT REVIEW

Recent findings in the molecular
biology of Salmonid Alphavirus

M. Brémont

Unité de Virologie et Immunologie Moléculaires, INRA, Université
Paris-Saclay, 78350, Jouy-en-Josas, France

Since almost four decades now, a disease called
Pancreas Disease (PD) affecting specifically
Atlantic salmon (*Salmo salar*) has been reported
in Scotland (Munro et al., 1984). The symptoms
associated with PD in salmon are a sudden loss
of appetite, lethargy and increased mortality.
The affected fish may have difficulty maintain-
ing position in the water due to muscle damage.
Later on a disease resembling PD but affect-
ing rainbow trout (*Oncorhynchus mykiss*) has
been described and named Sleeping Disease
(SD) (Anonymous, 1985; Boucher et al., 1994).
The etiologic agent responsible of PD/SD was
characterised 15 years ago as being the first fish
*Alphavirus*. To date alphaviruses inducing PD
and SD are termed Salmonid Alphavirus (SAV),
although this is not an official term. As for mam-
malian alphaviruses, the SAV genome consists
of a single-stranded positive-sense RNA mol-
ecule of about 12 kilobases (Kb) encoding two
polyproteins (Figure 1A), which after processing
through proteolytic cleavages produce the viral
mature products, the non-structural proteins
nsP1, nsP2, nsP3 and nsP4, which altogether
constitute the replicase complex and the major
structural proteins, the capsid (C) and the two
external glycoproteins (E2 and E1) (Figure 1B).
Compared to mammalian alphaviruses, SAV

exhibits several remarkable features like the
very short length of the 5' and 3' non coding
(NC) regions (approximately 20 versus 60 to 80
nucleotides for the 5' end and 90 versus >200 nu-
cleotides for the 3' end), the larger size of most
of the structural and non-structural proteins,
and the arthropod-independent way of trans-
mission to host. To date a considerable number
of complete or partial genomes from SAV field
isolates have been sequenced allowing a clas-
sification into 3 major subtypes (Hodneland
et al., 2005): SAV1 being the Salmon Pancreas
Disease Virus (SPDV) causing pancreas disease
in salmon in Ireland and Scotland, SAV2 the
Sleeping Disease Virus (SDV) causing sleeping
disease in freshwater rainbow trout and SAV3
SPDV causing pancreas disease in salmon and
marine rainbow trout in Norway. Infectious
SAV cDNA or SAV-derived replicons have now
been established in several laboratories which
have facilitated molecular biological studies on
SAV. Recent advances that have made use of
SAV replicons to express antigens of interest,
in both the development of SAV live vaccines
and the characterisation of factors involved
in the SAV pathogenicity for salmonids are
presented here.

<footnote>* Corresponding author's e-mail: michel.bremont@jouy.inra.fr</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1.** (A) Schematic representation of the SAV genome. The two-third 5' part encodes for the non-structural nsP1-4 proteins. The 3' third part encodes the structural, C-E3/E2-6K/E1 proteins. (B) Protein profile of sucrose-purified SAV on Coomassie blue stained SDS-PAGE.</figcaption></figure>

## Attempts to generate a SAV-derived infectious cDNA

As the genome of SAV is a single RNA molecule of positive polarity, RNA is infectious when transfected into the cells. However RNA cannot be directly manipulated so the use of a cDNA copy of the genome is a prerequisite to study the molecular biology of SAV. In the past, several approaches have been developed on mammalian alphavirus to generate infectious cDNA. The recovery of recombinant virus from cDNA is usually based on the transfection into cells of positive-stranded RNA generated by *in vitro* transcription from SP6- or T7-driven full-length viral cDNA constructs (Rice et al., 1987; Kuhn et al., 1991). When the RNA is transfected into the cells, it serves as a messenger RNA and new viral progeny is produced. For SAV, various similar constructs have been engineered however without success in recovering a replicative virus. Similarly SAV cDNA constructs under the control of the early eukaryotic cytosmegalovirus (CMV) promoter failed to produce a replicative SAV from transfected fish cells (Moriette, 2005). All the constructs tested are summarised in Figure 2. The 5'- and 3'-UTRs of SAV are the shortest known among alphaviruses

(Weston et al., 2002). As a result it has been hypothesised that SAV transcription initiation could be independent of a stem-loop structure in the genomic 5'- end (Moriette et al., 2006). However, *in silico* analysis suggested that SAV 5'-UTR might form two short stem-loops in the 5'-end (Karlsen et al., 2009). Thus it is not yet clear why in contrast to mammalian alphavirus *in vitro* transcribed SAV RNA genome and CMV-driven RNA transcription failed to succeed. To find a way of solving that blocking, SAV-derived mini-replicon expressing reporter genes like green fluorescent protein (GFP) or luciferase (LUC) in place of the SAV structural proteins were engineered (Moriette, 2005; Moriette et al., 2006) as previously described for mammalian alphaviruses (for a review see Frolov et al., 1996). When these replicons were transfected into fish cells neither GFP nor LUC expression nor non-structural proteins were detected in the transfected cells. However, when promoters were not directly fused to the 5' end of the minigenome (≥ 6 nucleotides), non-structural proteins were expressed in the transfected cells but not the reporter proteins. These observations suggested that to get expression of the non-structural proteins and reporter proteins
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Schematic representation on the different ways to engineer an infectious cDNA for salmonid alphavirus.</figcaption></figure>

as well the mini-replicon should be engineered
such that a spacer sequence has to be inserted
between the promoter and the 5' end of the
SAV genome and the RNA transcription must
start at the very first 5' end nucleotide. The best
way to fulfill these requirements was to fuse a
hammerhead ribozyme at the 5' end of the SAV
genome playing the role as spacer and allowing
the precise cleavage of the transcribed RNA at
the very first 5' nucleotide (Sarver et al., 1990).
That strategy was shown to be very efficient
since when these new constructs were trans-
fected into fish cells, the expression of luciferase
as well as GFP was detectable (Moriette et al.,
2006; Figure 3A and B). Since then a number
of similar SAV-derived replicons expressing
various antigens of interest instead of reporter
proteins have been published (Karlsen et al.,
2009; Wolf et al., 2013, 2014; Olsen et al., 2013;

Hikke et al., 2014; Abdullah et al., 2015; Guo
et al., 2015). Since SAV-derived replicons have
been shown to be functional, the next step has
been the recovery of replicative SAV. That has
been achieved by replacing in the SAV replicon
the genes encoding reporter proteins with the
gene encoding the SAV structural polypro-
tein. That was successfully accomplished for
SAV2 (Moriette et al., 2006) and later for SAV3
(Karlsen et al., 2010; Guo et al., 2014). For SAV2,
the recombinant virus (rSAV) generated was
shown to replicate in fish cells as well as the
wild-type SAV (wtSAV), however interestingly,
rSAV2 was totally attenuated in rainbow trout
when administered either by bath immersion
or even by injection, although high titers (10⁷
to 10⁸ PFU/mL) of replicative virus could be
recovered from sacrificed fish 2 to 3 weeks post
infection. Furthermore, A long-lasting (at least 7
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Functional SAV-derived replicons expressing either luciferase (A) or GFP (B). See main text for explanations.</figcaption></figure>

months) and complete protection was induced in the rSAV2-vaccinated trout challenged with the wtSAV2 by bath immersion and by injection as well. In addition, rSAV2-vaccinated fish were also protected from a challenge with the wtSAV3 (Moriette et al., 2006). In a recent study, an explanation for the attenuated phenotype of rSAV2 has been provided. It has been demonstrated that only a single amino acid change, A8V, in the SAV2 E2 glycoprotein was responsible for almost 90% of the rSAV2 attenuation (Mérou et al., 2013). Recently, a natural SAV variant of SAV3 which presented a single amino acid change P206S in the E2 glycoprotein was shown to replicate slower in cell culture but was as

virulent as the wild type SAV3 in fish although producing a less persistent infection in salmon (Karlsen et al., 2015).

In order to develop a safe live attenuated vaccine against Chikungunya, an alphavirus infecting humans and responsible of severe epidemics in the Indian Ocean islands, Hallengärd and collaborators (Hallengärd et al., 2014) have investigated whether the deletion of part of the Chikungunya encoding a small 6 K protein could lead to the generation of viable and attenuated virus. The resulting mutant was indeed attenuated and genetically stable. Furthermore this deletion mutant induced a strong T cell response
after a single immunisation in C57BL/6 mice. Based on that study, Guo and collaborators (Guo et al., 2014) tried to rescue a recombinant SAV3 lacking the 6K protein. Unfortunately, in contrast to Chikungunya virus, the 6K protein seems to be dispensable for the SAV3 viability. In conclusion, in the recent years molecular studies on SAV have exploded mainly due to the availability of reverse genetics systems which have been developed on SAV2 and SAV3. Finally to date only two vaccines have been developed, a live attenuated SAV2 vaccine which is not used in the field although administrable by bath immersion to trout (Moriette et al., 2006) and an inactivated SAV3 administrable by injection to salmon which is currently used in the field and was developed by a private company.

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