A naturally occurring substitution in the E2 protein of Salmonid alphavirus subtype 3 changes viral fitness
Introduction
The Salmon pancreas disease virus, commonly referred to as Salmonid alphavirus (SAV) (Weston et al., 2002), belongs to the Alphavirus genus in the Togaviridae family and has emerged as epizootics in farmed salmonids on at least six independent occasions following introductions of virus from a wild reservoir (Karlsen et al., 2014). This reservoir has been suggested to exist in or around the North Sea, and SAV has been found in wild caught fish species belonging to the order Pleuronectiformes from this area (Bruno et al., 2014, McCleary et al., 2014, Snow et al., 2010).
The 11.9 kB positive sense, single-stranded RNA genome of SAV is capped in the 5′ end, polyadenylated in the 3′ end and has two large open reading frames (ORFs) that encode the non-structural and structural proteins as two polyprotein precursors. The structural proteins consist of a capsid protein and the E3, E2, 6K/TF and E1 that together make up the glycoprotein spike that functions in receptor recognition (E2) and endosomal fusion (E1) (Firth et al., 2008, Weston et al., 2002). Viral transcription and replication is likely to occur in invaginations from endosomal membranes (Herath et al., 2012). The capsid protein locates mainly to the cytoplasm after translation, but contains a nuclear localization signal and can be detected in the nucleus of some cell types during replication (Karlsen et al., 2010b). The remaining part of the structural ORF is translated into the endoplasmatic reticulum as P62, 6K/TF and E1. P62 is cleaved by furin leading to the release of E3, which is not believed to be part of the SAV mature particle (Villoing et al., 2000a, Welsh et al., 2000). Budding probably occurs as a result of interactions between the glycoproteins and the capsid.
The cell and host tropism for SAV is not mapped to detail, but the RNA replication apparatus appears to be functional in a very wide range of species, cell types and temperatures (Olsen et al., 2013), suggesting that the structural proteins restrict host range and tissue tropism. Adaptations to new hosts therefore likely involve changes in these proteins.
Histopathological lesions caused by SAV in salmonids are found in pancreas, heart- and skeletal muscle, liver and central nervous system (McLoughlin and Graham, 2007). Presence of viral antigens in pancreas (Moriette et al., 2005, Taksdal et al., 2007, Villoing et al., 2000b) and skeletal muscle (Moriette et al., 2005) indicate that target cells exist in these organs. The virus is shed to the water from infected fish during viraemia (Andersen et al., 2010) and transmits to cohabitant fish (Nelson et al., 1995). Persistent infections are known (Andersen et al., 2007, Graham et al., 2010), but shedding of infectious particles is less likely to occur in this phase (Andersen et al., 2010).
SAV subtype 3 (SAV3) has been present in the Norwegian aquaculture industry since the mid 1980ies (Karlsen et al., 2014, Poppe et al., 1989). This epizootic is arguably the most comprehensive SAV epizootic in farmed fish to date and is spread within a cage through shedding to water from infected individuals. The reproduction number during this phase of transmission has been estimated to be between 1.0 and 2.9 (Tavornpanich et al., 2013). Transmission between farms is likely driven by a combination of direct horizontal transmission and human transport of fish (Karlsen et al., 2014, Viljugrein et al., 2009). SAV3 is still highly active in Norwegian fish farms, causing between 75 and 137 annually reported outbreaks between 2009 and 2013 (Hjeltnes, 2014). Two distinct genetic lineages have evolved after introduction from the wild reservoir, SAV3A and SAV3B. The lineages do not show any clear geographical pattern, and SAV3A is by far the more dominating in farmed salmon (Karlsen et al., 2014). A substitution from proline to serine in the E2 protein position 206 (E2206) occurred early in the SAV3A lineage. The substitution is interesting since it is one of few substitutions in E2 that separate the successful lineage 3A from the less successful lineage 3B, and since SAVH20/03, a strain belonging to 3A, changed back from a serine to a proline following serial passage in Chinook salmon embryo cells (CHSE-214) (Karlsen et al., 2006). These observations suggest that this substitution might be an adaptation to transmission in farmed populations of salmonids.
We modelled the 3D-structure of the SAVH20/03 P62 protein based on its homology to mammalian alphaviruses and found the spatial location of E2206 to be accessible in the B-domain. Recombinant SAV20/03 mutants were used to study the in vitro and in vivo effects of changing E2206 from proline to serine. Our results demonstrate that the substitution has reduced the in vitro fitness of SAV3 without affecting virulence or RNA replication in vivo dramatically. Its success in the ongoing SAV3 epizootic may instead be related to a change towards more rapid shedding dynamics.
Section snippets
Homology modelling and model validation
The extracellular domain of the P62 protein of SAVH20/02 was identified by alignment with the Chikungunya virus P62 sequence (PDB code 3N42) using T-coffee (Di Tommaso et al., 2011). The 3D structure of the SAVH20/03 P62 protein was predicted based on multiple-threading alignments and iterative template fragment assembly simulations as provided by the I-Tasser server (Roy et al., 2010, Zhang, 2008). The best fitting model was chosen by C-score and TM-score and imported into
Validation of a homology model of the SAVH20/03 P62 protein
The CHIKV derived structural template 3N40P was identified to be the most similar structure to the extracellular domain of the SAV P62 protein, sharing 25.7% sequence identity in this region. The best scoring model had a C-score of 0.87, a TM-score of 0.83 (±0.08) and an RMSD of 5.1 (±3.3)Å (Fig. 1a). Ramachandran plots of the SAV P62 protein showed 80.6% of positions in the most favourable region, 14.2% of positions in the allowed region, 2.7% of positions in the generously allowed region and
Discussion
The E2P206S substitution of the successful SAV3A lineage does not appear to have any dramatic impact on in vivo virulence or tissue tropism. It does however affect the function of the E2 protein, as evidenced by a slower rate of multiplication and reduced fitness in BF2-cells. The localization of position 206 in the E2 protein, exposed on the surface of the B-domain, combined with reduced in vitro fitness suggest that this change of fitness could be a result of altered receptor interaction (
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